1. society
2. crime
3. personal offense
4. homicide

© NYC OFFICE OF CHIEF MEDICAL EXAMINER
FORENSIC BIOLOGY PROTOCOLS FOR
FORENSIC MITOCHONDRIAL DNA ANALYSIS
Primary Approving Authority:
Mark A. Desire, Technical Leader – Mitochondrial DNA Operations
Table of Contents
Mitochondrial DNA Guidelines ..............................................................................4
A.
B.
C.
D.
General Procedures: ......................................................................................................... 4
Nomenclature ................................................................................................................... 5
Repeat Analysis of Samples ............................................................................................. 6
Batching and Duplication Guidelines .............................................................................. 7
Hair Evidence Examination ....................................................................................9
A.
B.
C.
Hair Evidence Examination ............................................................................................. 9
Mideo Macro/microscopic digital imaging system ........................................................ 10
Printing digital and mideo images for case file .............................................................. 12
Washing Hair for Mitochondrial or Nuclear DNA Testing ...............................13
A.
B.
C.
Demounting .................................................................................................................... 13
Washing the hair for mtDNA testing extraction ............................................................ 13
Washing the hair for nuclear DNA testing extraction .................................................... 14
Organic Extraction for Mitochondrial or Nuclear DNA Testing......................16
A.
B.
Extraction for Mitochondrial and Nuclear DNA testing ................................................ 16
Purification of DNA for Mitochondrial and Nuclear DNA testing ................................ 17
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Duplex Mitochondrial DNA PCR Amplification ................................................19
A.
B.
C.
Preparing the DNA aliquots for HVI-HVII amplification ............................................. 19
Amplification Setup ....................................................................................................... 20
Thermal Cycling ............................................................................................................. 22
Quantitation using Agilent 2100 Bioanalyzer .....................................................25
A.
B.
C.
Preparing the documentation:......................................................................................... 25
Preparing the samples..................................................................................................... 25
Procedure ........................................................................................................................ 25
Section A- Preparing the gel-dye mix ................................................................................... 26
Section B- Loading and running of the Agilent Bioanalyzer 2100 ...................................... 26
Section C- Data collection, analysis, electronic filing .......................................................... 28
Section D- Data Entry, Review, Filing, Rerun ..................................................................... 30
REVIEW ............................................................................................................................... 33
RERUNS ............................................................................................................................... 33
Mitochondrial DNA Linear Array Analysis........................................................34
A.
Hybridization.................................................................................................................. 34
B.
Photography ................................................................................................................... 38
C.
Interpretation of Results ................................................................................................. 39
D.
Sequence concordance ................................................................................................... 40
TROUBLESHOOTING ............................................................................................................ 43
Problem:
No bands on a Linear Array strip where there should be. ............................... 43
Problem:
High Background ............................................................................................. 44
EXO-SAP-IT Sample Cleanup .............................................................................46
Cycle-Sequencing ...................................................................................................48
SDS Cleanup ...........................................................................................................51
Centri-Sep Sample Filtration ................................................................................52
A.
B.
Procedure for Single Columns ....................................................................................... 52
Procedure for Centri-Sep 8 Strips .................................................................................. 53
ABI 3130xl Sequencing ..........................................................................................54
A.
Setting up a 3130xl Run ................................................................................................. 54
B.
Creating a Plate ID ......................................................................................................... 57
C.
Preparing the DNA Samples for Sequencing ................................................................. 58
D.
Placing the Plate onto the Autosampler (Linking and Unlinking Plate) ........................ 59
E.
Viewing Run Schedule and Starting Run ....................................................................... 60
F.
Water Wash and POP Change ........................................................................................ 62
3130xl Genetic Analyzer Troubleshooting ............................................................................... 63
Instrument Startup ................................................................................................................. 63
Spatial Calibration ................................................................................................................ 65
Spectral Calibration .............................................................................................................. 66
Run Performance .................................................................................................................. 67
Mitochondrial DNA Sequencing Analysis ...........................................................74
A.
B.
Transfer of the 3130xl run data into the master file ....................................................... 74
Sequence Analysis.......................................................................................................... 75
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C.
D.
E.
F.
Sequencher ..................................................................................................................... 77
File Output and Construction ......................................................................................... 83
Data Review ................................................................................................................... 83
Archiving the Sequencher Data ...................................................................................... 84
Sequence Nomenclature and Alignment ..............................................................87
A.
B.
Using Sequencher 4.9 ..................................................................................................... 87
Using Sequencher 4.1.4 .................................................................................................. 89
Editing Guidelines ..................................................................................................92
Interpretation Guidelines ......................................................................................93
GUIDELINES FOR CONTROLS ............................................................................................ 93
A.
Negative controls ........................................................................................................ 93
B.
Positive controls ......................................................................................................... 98
Guidelines for Reporting........................................................................................................... 99
A.
Linear array interpretation .......................................................................................... 99
B.
Sequencing: Reporting of Base Calls ....................................................................... 100
C.
Criteria for Mixture Recognition .............................................................................. 102
D.
Sequence Comparisons ............................................................................................. 102
Statistical Analysis ...............................................................................................105
Creation of a Casefile CD ....................................................................................110
References .............................................................................................................111
Appendix A –Oglionucleotide Primer Sequences .............................................114
Appendix B – Mitochondrial DNA Primer Locations ......................................116
Appendix C- Revised Cambridge Reference Sequence....................................118
Appendix D- Detailed CycSeq/3130xl Calculations ..........................................120
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Mitochondrial DNA Guidelines
PURPOSE: Guidelines for mitochondrial DNA testing to ensure clean laboratory practices,
unambiguous sample identification, and relevant control runs.
A. General Procedures:
To reduce the possible contamination in the laboratory that could occur: (i) between the
analyst and the samples (ii) from one sample to another, or (iii) from extraneous sources
of DNA within the laboratory.
1.
2.
3.
4.
5.
6.
7
8.
Lab coat, gloves, and mask, eye protection, and/or face shield, must always be
worn while in the exam and pre-amplification room. Lab coat, gloves, eye
protection must be worn in the post amplification area. All gowning must be
done in the vestibules of exam, pre-amp or post amp rooms.
Lab coats can be reused for a period of one week. Afterwards, they should be
thrown out. Masks/face shields can also be reused for a period of one week.
Goggles can be exposed to UV light in the Stratalinker to extend their time of use.
When working in the exam or pre-amplification laboratory, gloves must be rinsed
in 10% bleach before each procedure and in-between the handling of separate
samples.
Pipettes must be wiped down with 10% bleach before each procedure, and
between the pipetting of separate samples.
All hoods must be wiped down with 10% bleach before and after each procedure,
followed by a 70% Ethyl Alcohol rinse, and UV light, if available, should be
applied for 30 minutes before and following each procedure.
All racks, tube-openers and any other plastic implements (but not the pipettes)
must be exposed to UV light in the Stratalinker for a minimum of 30 minutes
before they can be used for amplification or extraction.
Any 96-well tube racks taken from the pre-amp room to the post-amp room must
be placed into the post-amp bleach bath, rinsed, and dried prior to being returned
to the pre-amp room.
All 1.5ml and 0.2ml tubes can be kept in plastic Nalgene boxes or comparable
containers, and should only be removed with bleached and dried gloves while
fully gowned. Prior to placement of tubes into these containers, the tubes used
for, washing, extraction and amplification must be exposed to UV light in the
Stratalinker for 30 minutes.
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9.
B.
Counters, sinks, refrigerator/freezer handles and door handles inside the
laboratory and gowning room should be wiped down with 10% bleach on a
monthly basis.
Nomenclature
The following are suggested naming conventions for use throughout the sample
processing. The goal of this nomenclature is to ensure that sample names are unique
identifiers.
1.
Samples re-extracted for the purposes of duplication (new cutting): The suffix :
”dup” will be added to the sample name to separately identify the re-extraction
sample from the original, and this suffix will be applied to these duplication
samples throughout the processing
2.
Samples reamplified in order to improve on the quality of the results or for other
purposes: The suffix “reamp” will be added to the sample name. If multiple
reamplifications are necessary, the numeral 1, 2, 3, etc. will be added to the suffix.
3.
At the 3130xl run step:
- The suffix “recyc” will be added to each sample name for samples that are resequenced (e.g. sample-recyc). If multiple re-cycle sequences are necessary,
the numeral 1, 2, 3, etc. will be added to the suffix.
- The suffix “conf” will be added to each sample name for samples that are resequenced to confirm sequence or length heteroplasmy (e.g. sample-conf). If
multiple confirmatory sequences are necessary, the numeral 1, 2, 3, etc. will
be added to the suffix.
- The suffix “reinj” will be added to each sample name for samples that are reinjected (e.g. sample-reinj). If multiple reinjections are necessary, the
numeral 1, 2, 3, etc. will be added to the suffix.
- The primer used will be added as suffix to each sample name. This suffix will
always be added last, e.g. sample-B4, sample-recyc-1-B4, sample-conf-2-B4,
sample-reinj-3-B4.
4.
Contig name:
- A contig name should be: FBYY-12345-HVI, or FBYY-12345-HVII, or
FBYY-12345-HVI dup, FBYY-12345-HVII dup
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-
5.
C.
In certain cases more than one sample will be typed and identifiers could be
added after FBYY-12345, e.g. FBYY-12345(V)-HVI, FBYY-12345-PM7HVI, FBYY-12345-PM1-HVI, FBYY-12345-Q1-HVI, FBYY-12345-Q2-HVI
Sequencher ID
- The sequencher ID of an analyzed run will be identical to that run ID, e.g.
SYY-123.
- The sequencher ID of a case will be identical to the FB case number, e.g.
FBYY-12345.
- The sequencher ID for a Missing person case will be the FB# space MP (e.g.
FBYY-12345 MP)
Repeat Analysis of Samples
Repeat testing of a sample can start at different stages, as listed below. Appropriate
controls must be used.
1.
2.
3.
Extraction stage: A new extraction negative control must be run.
Amplification stage: New amplification negative and positive control must be
included. The extraction negative control does not need to be repeated if it
previously passed.
Cycle sequencing: Positive and negative controls must be tested for each primer
used. The original extraction negative does not have to be repeated if it passed for
all needed sequences. The original amplification negative does not have to be
repeated if it passed for all needed sequences; a cycle sequencing negative (cAN)
should then be used (20 µl H2O) for each primer used. The original positive
control should be used and suffixed recyc for each primer used; however, any
positive control can also be used as long as the contig of interest can be built with
that positive control. Note that if a sample needs to be re-sequenced with a primer
because the positive control at that primer failed, then every control or sample in
that run needs to be re-sequenced with that primer.
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D.
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Batching and Duplication Guidelines
Duplication of samples is only necessary from when samples are batched.
Exemplar samples batched and extracted for nuclear DNA may be duplicated with a
second nuclear DNA extraction and STR typing.
For mtDNA, duplication of a given sample can be accomplished by running one
informative primer for that sample in either HVI or HVII.
1.
2.
Evidence samples
a)
Batching of evidence samples will be allowed at all steps of mtDNA
analysis including the DNA extraction stage. When batching evidence
samples at the extraction stage, a maximum number of five samples can be
batched. Duplication at the extraction level can be done for case-related
reasons (see supervisor).
b)
Identical mtDNA profiles involving at least one evidence sample (two
evidence samples or one evidence sample plus an exemplar) within a case
are considered duplicated for the evidence sample.
c)
Duplication of sample at the quantification level is not required. Evidence
samples may be duplicated if they do not match any other sample in the
case at the discretion of a supervisor.
d)
Duplication of evidence samples may begin at the amplification or cycle
sequencing steps if there is no additional evidence material for extraction
or amplification, respectively.
Exemplar samples
a)
b)
c)
Batching of exemplar samples from different cases will be allowed at all
steps of mtDNA analysis including the DNA extraction stage.
HVI-HVII amplification and sequencing of exemplar samples from the
same case (e.g. family members, duplication samples) should be
performed at least once separately.
Suspect exemplars will be duplicated if that sample matches an evidence
sample.
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d)
e)
f)
3.
Victim exemplars will be duplicated if they do not match any other sample
in the case and if that exclusion is informative.
Missing Persons’ exemplars and unidentified remains do not need to be
duplicated.
Any exemplar may be duplicated for case related reasons or to streamline
testing.
Exemplar with Evidence samples
With the exception of quantification evidence and exemplar samples must always
be tested separately in time and/or space. Batching of evidence with exemplar
samples is allowed during the DNA quantitation step. Batching of evidence with
exemplar samples is also allowed during Agilent analysis provided that sample
aliquots are done on each sample type (evidence or exemplar) at separate times.
Quantification steps do not need to be duplicated.
Revision History:
July 24, 2010 – Initial version of procedure.
February 28, 2011 – Removed “product gel” from Paragraph D.3.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
April 1, 2014 – Modified section D (Batching and Duplication Guidelines). Batching of evidence in now allowable at all
steps of mDNA analysis. Policy for duplication of evidence has been modified to be at the discretion of the supervisor
if sample results are not duplicated in the case.
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HAIR EVIDENCE EXAMINATION
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Hair Evidence Examination
General Guidelines
Refer to Evidence and Case Management Manual for general laboratory guidelines for evidence
examination, note-taking, itemization, and preparation for evidence examination.
A. Hair Evidence Examination
1.
Record all packaging documentation and open packaging in a dead-air hood
(preferred) or comparable (e.g. enclosed space).
2.
Document the hair examination. Note the hair approximate length and whether or
not the hair is mounted.
If hair is <1 cm in length, see supervisor. If the hair will be consumed
indicate in case notes that the sample will be consumed for testing and
proceed.
For hairs that are loose, proceed with step 5. For hairs that are mounted, proceed
with demounting (See “Washing Hairs for Mitochondrial and Nuclear Testing”,
part A) and then return to step 5.
Note: It is at the analyst’s discretion to photo document hair mounted on a
slide at this step. In this case follow step 5 below prior to demounting hair.
Take a picture of the full hair:
• Digital or Mideo picture can be made.
• If the hair is unmounted, it can be placed in a weigh boat.
• Place hair on appropriate background for photo documentation. Brown,
black, or darker colored hairs should be placed on a white sheet of clean
paper. Blonde, white or light colored hairs should be placed on a darker
background.
• Take a digital/Mideo photograph of the full hair, including a
ruler/measurement in the frame. For digital pictures, be sure the digital
camera is set to Macro (flower) and the flash is off before taking the picture.
For Mideo pictures, see Mideo Macro/Microscopic Digital Imaging System
below (see part B).
i.
Save /export pictures as the LIMS attachments.
ii. Print digital and Mideo images for case file (see part C).
3.
4.
5.
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B. Mideo Macro/microscopic digital imaging system
1.
Make sure the computer is on and all of the proper cables are connected between
the Firewire camera and the computer.
2.
Double-click on the desktop shortcut EZDocPlus.
3.
The main program screen will appear. Click on “Camera” and select “Micro Cam
M”. The QCam Microcam Control Panel will appear.
4.
Make sure at this point that the stereo microscope is on, the light source is active,
and the specimen is in focus. When viewing solid, dark objects, it is best to use
the ocular light ring to illuminate the sample. When viewing slides or thin tissue
samples, use the direct light from the lamp base of the microscope.
5.
On the Microcam Control Panel (shown to the right), perform the following:
a.
b.
c.
d.
e.
Click the Live button.
Adjust the binning so that the setting is 3 for
both the horizontal and vertical.
Check the Flip Image box.
Click the Auto Exposure button.
Adjust the intensity of the light and click
Auto Exposure if the image is too bright or
too dark.
NOTE: If the background color is not white or
off white, place a sheet of paper in view of the
lens and click on the White Balance button.
Repeat step 5d once completed.
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6.
Once the image on the screen is in focus and there is proper contrast, click the
Grab button on the control panel. This will freeze the image on the screen.
7.
Under the Tools menu, select Overlays. Once the panel opens up, perform the
following:
a.
b.
c.
d.
e.
Click on the Measure tab.
Click the Load Calibration button.
Select the calibration based on the current microscope magnification level.
FOR LINEAR OBJECTS
1)
Click on the Length button.
2)
Select any of the length tools to measure the length of the imaged
object.
FOR NON-LINEAR OBJECTS
1)
Click on the Multilength button
2)
Trace the non-linear object length by left-clicking the mouse at
desired turns and corners. Hit “Enter” on the keyboard when
finished.
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8.
Save the image by going to the File menu and selecting Export Image.
9.
Save image with case identifying name (e.g. FB07-04117 Item 1A-1).
10.
Import the saved image into the LIMS as an attachment for the evidence item.
C. Printing digital and mideo images for case file
1.
2.
3.
4.
5.
Open Microsoft PowerPoint.
Go to File menu and select Page Set-up. Change slide orientation from landscape
to portrait.
Import pictures
Add Sample ID. Add comments if needed.
Save as FB# in Photo Archives
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Washing Hair for Mitochondrial or Nuclear DNA Testing
PURPOSE: To prepare hairs for DNA extraction.
A. Demounting
1.
If the hair is loose, then proceed to the appropriate hair washing procedure. If a
“possible root” is observed, the sample should be cut and washed for nuclear
DNA testing extraction (see part C).
2.
If the hair is mounted:
i.
Process only one mounted slide at a time.
ii.
Turn on the heat plate and adjust the heat dial between 100-110oC. Place
the slide on a heat plate until the mountant softens and using forceps
remove the cover slip. The mountant softens quickly and hairs will scorch
if left on the heat plate too long.
iii.
The hair will be attached to either the coverslip or the slide. Remove hair
and place into a xylene bath for up to 5 minutes or until the mountant
completely dissolves. Hairs and slides/coverslip containing hairs can be
kept in the xylene bath for longer than 5 minutes if necessary.
iv.
Using clean forceps, carefully remove the hair from the xylene bath
v.
It is at the discretion of the analyst to make a picture of the full hair at this
time.
vi.
Proceed to the appropriate hair washing procedure.
B. Washing the hair for mtDNA testing extraction
1.
2.
3.
4.
Using forceps and a scalpel cut a 2 cm region of the hair or hair shaft. A picture
of the cutting should be taken at this time. If the hair is also to be tested for
nuclear DNA, the mitochondrial DNA cutting should be away from the root.
Place the unused portion of the hair onto the backing of a post-it note and return
to the packaging
If “possible tissue” attached to hair is observed, see your supervisor. In some
cases the hair will not be washed, proceed to step 11 and enter N/A as
TergAZyme and Saline lot #.
Prepare 5% TergAZyme solution by adding 15ml of GIBCO water to 0.75g of
TergAZyme. Mix well. Record TergAZyme lot #.
Using clean forceps, place the hair fragment cutting into a 1.5 ml tube with 1 ml
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5.
6.
7.
8.
9.
10.
11.
12.
13.
of 5% TergAZyme solution. Vortex the tube for 1 minute at high speed, and
place into the sonicator for 15 minutes. After sonication, vortex the sample again
for 1 minute at high speed.
Prepare a 50 ml Falcon tube and filter cup. Label the tube and filter cup tab with
the sample name. Pre-wet the filter cup membrane with 1 ml of Gibco dH2O.
Remove the hair from the TergAZyme with clean forceps, and place the hair into
the filter cup in the center of the membrane.
Wash the hair with 1 ml of Gibco dH2O. Allow the liquid to pass through the
filter.
Wash the hair with 1 ml of 0.85% saline. Allow the liquid to pass through the
filter. Record Saline Lot #.
Wash the hair with 1 ml of 100% ethanol. Allow the liquid to pass through the
filter.
Remove the filter cup containing the hair and place on a Kimwipe to let the
ethanol evaporate. Once the filter membrane is dry, the hair will be dry as well.
Transfer the cut hair fragment to the bottom of a clean 1.5 ml tube. Label the tube.
Store the tube containing the hair fragment in the appropriate “To Be Extracted”
cryobox in the pre-amplification laboratory freezer.
Proceed to Mitochondrial extraction for mitochondrial DNA testing procedure.
C. Washing the hair for nuclear DNA testing extraction
1.
2.
3.
4.
5.
For nuclear DNA extractions, using forceps and a scalpel cut up to 1.5 cm of the
proximal region of the hair, including the root. Place the unused portion of the
hair onto the backing of a post-it note and return to the packaging. A picture of
the root should be taken at this time.
If “possible tissue” attached to hair is observed, see your supervisor. In some
cases the hair will not be washed, proceed to step 11 and enter N/A as Saline
lot #.
Prepare a 50 ml Falcon tube and filter cup set by labeling the tube and filter cup
tab with the sample name. Pre-wet the filter cup membrane with 1 ml of 0.85%
saline. Document Saline lot #.
Using clean forceps, place the cut hair into the filter cup in the center of the
membrane.
Wash the hair with 1 ml of 0.85% saline. Allow the liquid to pass through the
filter. Repeat that step.
Wash the hair with 1 ml of 100% ethanol. Allow the liquid to pass through the
filter.
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6.
7.
8.
9.
10.
Remove the filter cup containing the hair and place on a Kimwipe to let the
ethanol evaporate. Once the filter membrane is dry, the hair will be dry as well.
Transfer the cut hair fragment to the bottom of a clean 1.5 ml tube. Label the tube.
Store the tube containing the hair fragment in the appropriate “To Be Extracted”
cryobox in the pre-amplification laboratory freezer.
Within the LIMS system, indicate the cutting was made on the evidence item and
schedule the appropriate DNA extraction procedure using the sample creation
wizard.
Proceed to Mitochondrial extraction for nuclear DNA testing procedure.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
ORGANIC EXTRACTION FOR MITOCHONDRIAL OR NUCLEAR DNA TESTING
DATE EFFECTIVE
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Organic Extraction for Mitochondrial or Nuclear DNA
Testing
Refer to the current Protocols for Forensic STR Analysis manual for extraction, quantitation,
amplification, and STR procedures currently on-line for other Nuclear DNA Operations.
PURPOSE:
A.
To isolate nuclear or mitochondrial DNA from the hair using an enzymatic
digestion of the hair followed by an organic extraction.
Extraction for Mitochondrial and Nuclear DNA testing
1.
2.
3.
4.
5.
6.
7.
Prepare hair for digestion by removing the appropriate microcentrifuge tube from
the “To Be Extracted” cryobox. Record the Organic Extraction documentation.
Prepare the incubation solution in a 1.5ml tube using the following table. Label
this tube with the extraction date and time as ENEGDDMMYY-HHMM.
Reagents
1 hair + extraction negative
Proteinase K (20mg/ml)
30 :l (15*2)
DTT (1M)
75 :l (37.5*2)
20% SDS
7.5 :l (3.75*2)
Organic Extraction Buffer
188 :l (94*2)
When extracting clumps of hair, or multiple hairs together, the total volume of the
incubation solution can be increased 2- to 10-fold, to accommodate the size of the
sample. Adjust the reagent volumes to accommodate these changes. Be sure to
record such volume changes in the documentation.
Have the extraction tube set-up witnessed.
Aliquot 150 :l of the incubation solution into the 1.5ml tube containing the hair
and leave the remaining solution in the original 1.5ml tube as the negative control.
Incubate samples for 30 min. in a 1400 rpm shaker at 56EC. Record the number
of the thermal mixer , the thermal mixer temperature setting and actual
temperature in the documentation.
After 30 min., hairs should be dissolved. If not, incubate for a total of 1-2 hours.
If hairs have not dissolved, add 1:l of 1M DTT and incubate overnight. Be sure
to record this on the extraction documentation, if performed. Hairs and control
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
ORGANIC EXTRACTION FOR MITOCHONDRIAL OR NUCLEAR DNA TESTING
DATE EFFECTIVE
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8.
9.
B.
samples should be both treated the same way. After overnight incubation, record
the thermal mixer setting and temperature in the documentation.
When the hair sample is completely dissolved, proceed with the extract to the
purification step (see part B)
The hair sample might not completely digest even after the overnight incubation.
If the hair is chemically treated, straightened, or dyed, it might resist digestion.
The incubation process might remove the pigment or coloring from a hair and
leave it opaque. If this happens, record this observation in the documentation.
Centrifuge the sample for 3-5 minutes at full speed. Collect the supernatant
(extract) in a new tube, carefully without disturbing the pellet. Add the suffix “–
R” to the sample name and label on the original tube containing the hair remain
(pellet). Hair remains will be stored with the other sample. Proceed to purification
step with the extract (see part B).
Purification of DNA for Mitochondrial and Nuclear DNA testing
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
During the incubation, prepare and label for each sample: one Eppendorf Heavy
Phase Lock Gel (PLG) tube, one microcon filter, three microcon collection tubes,
and one 1.5 ml tube for final extract. PLG tubes can be centrifuged for 30
seconds at maximum speed prior to sample addition.
After incubation, have the purification tube set-up witnessed.
Transfer each extracted sample to appropriate labeled PLG tube. PLG tubes make
the phase separation between organic and aqueous layers of an organic extraction
easier. To each PLG tube add an equal volume of Phenol: Chloroform: Isoamyl
Alcohol (25:24:1 PCIA). The PCIA volume to be added should be 150µl unless
the extraction volume has been increased in step A3. PCIA is an irritant that is
toxic. Its use should be confined to a certified fume hood. Gloves and a
mask should be worn.
Shake or briefly vortex the tube to achieve a milky emulsion.
Centrifuge the tube in a microcentrifuge for 2 minutes at maximum speed.
Insert Microcon DNA Fast Flow filter cup (blue) into labeled microcon tubes for
each sample.
Prepare the Microcon concentrator by adding 100 :l of TE-4 to the filter side (top)
of the concentrator.
Transfer the aqueous phase (top layer) from the PLG tube to the prepared
Microcon concentrator. Do not disturb the PLG layer. Discard the PLG tube
containing the organic layer into the organic waste bottle in the fume hood.
Spin the Microcon concentrator for 25 minutes at 500 rcf.
Transfer the Microcon filter cup into a new labeled Microcon tube and add 400 :l
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
ORGANIC EXTRACTION FOR MITOCHONDRIAL OR NUCLEAR DNA TESTING
DATE EFFECTIVE
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11.
12.
13.
of TE-4 to the filter side (top) of the concentrator.
Spin again at 500 rcf for 20 minutes. After this spin, if liquid is still observed on
the membrane, spin again at 500 rcf for an additional 6 minutes. After this spin, if
liquid is still observed on the membrane, continue spinning for a longer time.
Add 20 :l of TE-4 to the filter side (top) of the concentrator.
Invert the blue concentrator cup and place into appropriate microcon collection
tubes. Spin at 1000 rcf for 3 minutes to collect samples.
• For mitochondrial DNA testing,
o Using a pipetter, measure volume collected and record it.
o Transfer samples to a 1.5 ml microcentrifuge tube for storage.
o Adjust samples volume to 50 :l using TE-4 record these volumes.
o Proceed with HVI-HVII amplification with 20µl of samples.
• For nuclear DNA testing,
o Using a pipetter, measure the volume collected
o The volume should be close to 20ul, in control and hair samples. If the
volume is >30µl, prepare a new microcon filter and tube (see part 6 above)
and spin at 500 rcf, control and hair samples, for an additional 6 minutes.
After this spin, if liquid is still observed on the membrane, continue
spinning for a longer time.
o Measure the final volume collected and record it.
o Transfer samples to a 1.5 ml microcentrifuge tube for storage
o Send 2.5 µL of samples (neat) for nuclear quantification. If quantitation
results show an insufficient amount of nuclear DNA for STR testing, the
extract may then be used for mtDNA analysis.
Revision History:
July 24, 2010 – Initial version of procedure.
November 4, 2010 – Added instruction for thermal mixer documentation.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
December 31, 2012 – YM100 microcons were discontinued by the manufacturer. The manufacturer is now producing the
DNA Fast Flow Microcons. All references to the YM100’s have been revised. Spin times in Section B, Steps 11 and
13 have been revised for the new microcons.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
DATE EFFECTIVE
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Duplex Mitochondrial DNA PCR Amplification
PURPOSE:
To increase the amount of available mtDNA for the purposes of analysis, by
performing an in vitro replication of template DNA using oligonucleotide
primers, thermostable DNA polymerase and deoxynucleoside triphosphate bases
(dNTPs) within a thermal cycler.
PROCEDURE:
A positive control, an amplification negative, and an extraction negative control (if applicable)
should be included with each batch of samples being amplified to demonstrate procedural
integrity. The positive control is a laboratory grade cell line, for which the mtDNA type is
known.
Follow the mtDNA pre-amplification guidelines for handling the tubes and cleaning of the work
surfaces. The following steps have to be performed in the appropriate dedicated areas. Evidence
samples and exemplar samples should not be handled at the same time.
A.
Preparing the DNA aliquots for HVI-HVII amplification
o
Amplification can be performed with either Roche or Homebrew reagents.
Homebrew reagents should be used for Missing Person’s testing only.
o
When amplifying extracts which have nuclear DNA quantification data, the target
amount of extract to be amplified is:
100 pg when using Roche reagents
500 pg when using Homebrew reagents
o
When amplifying samples that have not been quantified (e.g., hair shaft samples),
use 20ul of the extract and Roche reagents, only.
o
Table I refers to the preparation of the control samples for the amplification.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
DATE EFFECTIVE
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Table I – Control samples for amplification.
Sample
DNA (extract)
TE-4
HL60 Positive Control DNA (100 pg/20 :l)
20 :l
---
Amplification Negative Control
---
20 :l
Extraction Negative Control,(s) when sample 20 :l
amplified neat
Extraction Negative Control (s) when a
dilution/concentration of sample extract is
amplified
B.
---
Submit 20 :l of the extraction
negative at the same
dilution/concentration factor or
more concentrated than the
sample
Amplification Setup
1.
For each amplification set, record the lot numbers and samples in the
documentation. Label 0.2 ml PCR reaction tubes with sample label name and
with date and time for the positive and negative controls.
2.
If samples require dilution, prepare the aliquots in UV’ed 1.5mL tubes, and place
the neat samples back into storage.
3.
Master mix preparation:
a)
For amplification using Roche reagents:
•
Prepare a Master Mix with Reaction Mix and Primer Mix, The
following calculations are used:
o
Reaction Mix: number of samples N x 20 :l Reaction Mix
= __ :l
o
Primer Mix: number of samples N x 10 :l Primer Mix =
__ :l
Note: For ≤6 samples, use N, for ≥ 6 samples, use N+1. To
save on reagents, individual aliquots of Reaction and
Primer Mix can be made.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
DATE EFFECTIVE
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o
o
o
b)
Add Reaction Mix and Primer Mix together to prepare
Master Mix.
Vortex the Master Mix and centrifuge briefly.
Aliquot 30 :l of the Master Mix into the bottom of each
labeled 0.2 ml reaction tube.
For amplification with Homebrew reagents:
•
Prepare Reaction Mix and Primer Mix master mixes according to
Homebrew amplification documentation. The following amounts
of reagents per sample are used:
Reaction Mix
Reagents
Irradiated dH2O
GeneAMP 10X
PCR Buffer
2.5mM dNTPs
25mM MgCl2
5U/µL AmpliTAQ
Gold DNA
Polymerase
Total Reaction
Mix volume per
sample
Volume
per sample
3.7 µl
5 µl
Primer Mix
Reagents
Irradiated dH2O
10µM HVIF
Volume
per sample
6 µl
1 µl
4 µl
4.8 µl
10µM HVIR
10µM HVIIF
1 µl
1 µl
2.5 µl
10µM HVIIR
1 µl
20 µl
Total Primer Mix
volume per sample
10 µl
The following calculations are used:
o
Reaction Mix: number of samples + 1 (N + 1) x Reaction
Mix Reagent Amount = __ :l
o
Primer Mix: number of samples + 1 (N + 1) x Primer Mix
Reagent Amount = __ :l
•
•
Vortex the Reaction and Primer Mix and centrifuge briefly
Aliquot 20 :l of the Reaction Mix and 10ul of the primer mix into
the bottom of each labeled 0.2 ml reaction tube.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
C.
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4.
Add samples into the 0.2ml tubes. Use a sterile filter pipette tip for each sample
addition. Open only one tube at a time for sample addition. The final
aqueous volume in the PCR reaction mix tube will be 50 :l. Transfer the
appropriate volume of target DNA or TE-4 to each respective sample tube. After
the addition of the DNA, cap each sample before proceeding to the next tube. If
necessary, spin down the tubes at 1000 rcf for a few seconds.
5.
When finished, place the rack with the 0.2ml tubes in the pre-amp room
dumbwaiter. Send the samples up to the post-amp room.
Thermal Cycling
1.
Turn on the Perkin Elmer 9700 Thermal Cycler.
2.
Use the following settings to amplify the samples
9700 Thermal Cycler
User: mtDNA
File: lamtdna
The amplification file is as followsSoak at 94EC for 14 minutes
- Denature 92EC for 15 seconds
34 cycles:
- Anneal at 59EC for 30 seconds
- Extend at 72EC for 30 seconds
Incubation at 72EC for 10 minutes
Storage soak at 4EC indefinitely
3.
Place the tubes in the tray in the heat block, slide the heated lid over the tubes,
and fasten the lid by pulling the handle forward. Place the microtube rack used to
set up the samples for PCR in the post-amp room bleach bath
4.
Start the run by performing the following steps:
a.
b.
c.
The main menu options are RUN CREATE EDIT UTIL USER. To
select an option, press the F key directly under that menu option.
Verify that the user is set to “mtDNA” if not, select the USER option (F5)
to display the “Select User Name” screen.
Use the circular arrow pad to highlight “mtDNA.” Select the ACCEPT
option (F1).
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
DATE EFFECTIVE
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d.
Press the RUN button (F1) and select the “lamtdna” file.
e.
Verify that the reaction volume is set to 50 :l and the ramp speed is set to
9600 (very important).
f.
If all is correct, select the START option (F1).
The run will start when the heated cover reaches 103EC. The screen will
then display a flow chart of the run conditions. A flashing line indicates
the step being performed, hold time is counted down. Cycle number is
indicated at the top of the screen, counting up.
g.
Be sure to record the use of the thermal cycler in the documentation under
the appropriate instrument name. .
h.
Upon completion of the amplification, press the STOP button repeatedly
until the “End of Run” screen is displayed, and remove your samples.
Select the EXIT option (F5). Wipe any condensation from the heat block
with a Kimwipe and pull the lid closed to prevent dust from collecting on
the heat block. Turn the instrument off.
i.
After removing your samples, place them in the appropriate 2-8 °C
refrigerator for storage. Samples should be separated according to sample
type (exemplar, evidence, or quality control). Record the date and time of
when samples were amplified on the cover of the 0.2 mL PCR storage
box.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
DUPLEX MITOCHONDRIAL DNA PCR AMPLIFICATION
DATE EFFECTIVE
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IMPORTANT:
Turn instruments off ONLY when the Main Menu is displayed, otherwise there will be a Power
Failure message the next time the instrument is turned on. It will prompt you to review the run
history. Unless you have reason to believe that there was indeed a power failure, this is not
necessary. Instead, press the STOP button repeatedly until the Main Menu appears.
In case of a real power failure the 9700 thermal cycler will automatically resume the run if the
power outage did not last more than 18 hours. The Uninterruptible Power Supply (UPS) present
in the amplification room will power the thermal cyclers for about 2-3 hours in the case of a total
power outage. The history file contains the information at which stage of the cycling process the
instrument stopped. Consult with the QA team and/or the Technical Leader on how to proceed.
Revision History:
July 24, 2010 – Initial version of procedure.
December 22, 2010 – Added procedure for Homebrew amplification. Changed title of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
QUANTITATION USING AGILENT 2100 BIOANALYZER
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Quantitation using Agilent 2100 Bioanalyzer
PURPOSE- To quantify the amplified product of the mitochondrial hypervariable regions I
and II, in order to establish the input of DNA for linear array analysis and/or cycle sequencing.
The DNA 1000 assay is capable of analyzing amplified DNA fragments in the range of 25-1000
bp, and in the concentration range of 0.5-20 ng/uL.
A. Preparing the documentation:
a. Prepare a new Agilent Test Batch, and select samples for analysis
b. Exemplar samples and positive controls should be run at 2-fold (d2) and 5-fold
(d5) dilutions. Hair and evidence samples should be run d1 (neat) and d2.
Negative controls should be run d1 (neat). Prepare output samples accordingly.
c. Exemplar and evidence samples may be quantitated on the same Agilent run;
however, they must be aliquotted for quantitation separately.
d. Prepare the plate record, and manually link the samples into the associated plate
wells.
e. Download the plate record, and confirm the .CSV file is saved in the appropriate
network location.
B. Preparing the samples
a. Follow the documentation to prepare dilutions. Add H2O first in all tubes where
needed.
b. Vortex and centrifuge tubes between serial dilutions
c. Aliquot all volumes less than 2 µl using a 2 µl pipette.
d. When pipetting sample for dilution or quantitation, pick up from the top of the
solution (directly on the meniscus) to avoid carrying sample on the outside of the
tip.
e. Use 1µl for quantification. Vortex and centrifuge every tube before use.
C. Procedure
a. If the Gel-Dye mix is not prepared, proceed to SECTION A.
b. If the Gel-Dye mix is already prepared, proceed to SECTION B.
c. For analysis of data only, proceed to SECTION C.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
QUANTITATION USING AGILENT 2100 BIOANALYZER
DATE EFFECTIVE
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POWDER-FREE GLOVES ARE REQUIRED FOR THE HANDLING OF AGILENT
CHIPS. POWDER FROM LATEX GLOVES COULD CLOG THE MICRO-CHANNELS
ON A CHIP.
Section A- Preparing the gel-dye mix
1.
Allow the DNA dye concentrate (blue tube) and the DNA gel matrix (red tube) to come
to 37°C in the heat block.
2.
ALWAYS PROTECT THE DYE CONCENTRATE FROM THE LIGHT. Vortex the
DNA dye concentrate (blue tube) and spin down. Add 25 :l of the dye concentrate (blue
tube) to the DNA gel matrix vial (red tube).
3.
Vortex the mixture for 10 seconds to ensure complete mixing, and transfer the entire
mixture to the top receptacle of a spin filter.
4.
Centrifuge for 15 minutes at 6000 rpm. Discard the filter and label the gel-dye mix tube
with the lot numbers of the DNA dye concentrate, the DNA gel matrix, and your initials
and the date.
5.
One tube of gel-dye mix is enough for 10 runs, and will last for 4 weeks. Discard the geldye mix 4 weeks after the date of preparation. Protect the gel-dye mix from light, and
store at 4EC. Record the creation of the gel-dye mixture in the LIMS.
Section B- Loading and running of the Agilent Bioanalyzer 2100
2100 Expert System Setting (left inside of the window, click on system) are saved by default as:
1. Data Files Name: serial number, data, time are checked.
2. Data Files directory: “Create Daily subdirectories” is checked.
3. Data File format: “Binary format” is checked.
4. Nothing is checked in “Run and Results”, “Auto Export,” and, “Default Export
Directories”.
ALL PIPETTING INTO THE CHIP MUST BE DONE DIRECTLY ON THE GLASS AT
THE BOTTOM OF THE WELL, NEVER ON THE SIDES OF THE WELL.
1.
Allow the gel-dye mix to equilibrate to room temperature.
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QUANTITATION USING AGILENT 2100 BIOANALYZER
DATE EFFECTIVE
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2.
Have a witness check samples and worksheet. Record the run name, the lot#, analyst,
date, time, and the Agilent machine, in the documentation. Open a new DNA chip
3.
Pipette 9 :l of gel-dye mix into the bottom of the well marked G. Make sure there are no
bubbles, if any use a 1µl pipette tip to remove them. Place it in the priming station, and
fill out usage log. Make sure the base plate of the station is set to position C, and the clip
on the syringe trigger is set to the lowest position. Make sure the syringe piston is pulled
back to the 1 ml mark, and close the lid of the priming station. (Listen for the “click.”)
4.
Grab the syringe with your index fingers under the fins on the syringe body and thumbs
on the plunger. Swiftly and steadily, press down on the plunger until it locks under the
silver trigger. Make sure your thumbs are not in the way of the trigger lock or it will not
work. Let the chip pressurize for 60 seconds.
5.
Release the syringe with the trigger, and make sure the syringe comes back to 0.3-0.4 ml.
Wait for 5 seconds, pull slowly the syringe back to 1 ml, and open the chip priming
station. Turn the chip over and inspect the capillaries for proper filling.
6.
Pipette 9 :l of gel-dye mix into the two wells marked G. Make sure there are no bubbles,
if any use a 1µ tips to remove them.
7.
Vortex and spin down the DNA marker (green tube), and pipette 5 :l of marker into each
of the 12 sample wells and ladder well. Each well must be filled, even if it will not be
used.
8.
Vortex and spin down the DNA ladder (yellow tube), and pipette 1 :l of ladder into the
lower right well, marked with the ladder symbol.
9.
Add 1 µL of amplified DNA to each well. If a well is not used, add 1 :l of dH2O into the
well.
10.
Place the chip in the IKA vortexer and vortex at ~2200 rpm for 60 seconds.
11.
Run chips within 5 minutes.
12.
Start the collection software by clicking on the symbol on the desktop.
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QUANTITATION USING AGILENT 2100 BIOANALYZER
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13.
Click on “instrument” on the left panel and place the cursor in one cell of the Sample
Name column, click on the right button of the mouse, select import, choose the .CSV text
file that was created for the run (see A7). The software might give a warning “failed to
import the text file.” Ignore, and press OK.
14.
Once the machine is highlighted in the upper left-hand corner of the screen, open the lid.
The icon should now show the lid open as well. Insert the DNA 1000 chip and carefully
close the lid. The machine icon will now change to a blue chip on the screen. Make sure
the “Assay Class” in the “Assay Details” panel (middle right panel) is “DNA 1000”. If
the assay class is different than DNA 1000 see a supervisor before starting the run.
15.
Adjust the sample # in the Data Acquisition Parameters field, if necessary.
16.
Click on the START button.
The run will begin with a pre-heat and should take approximately 35-40 minutes for a full
chip.
17.
The ladder sample will process first. It is a good idea to monitor this sample to make
sure the upper and lower markers come out correctly (15 bp and 1500 bp).
18.
Immediately after the run (less than 5 minutes), remove the sample chip. The electrodes
need to be cleaned with the clear electrode cleaner chip within 5 minutes after the run.
To do this, begin by filling one of the large wells with 350 :l of deionized water. Place
the electrode cleaner in the Agilent 2100 Bioanalyzer and close the lid for 10 seconds
(and not more than 10s). Open the lid, remove the electrode cleaner chip, and let the pin
set dry for another 10 seconds (and not more than 10s), then close the lid. Drain and dry
the electrode cleaner chip.
Section C- Data collection, analysis, electronic filing
When the Bioanalyzer 2100 run is complete, go to the Data & Assay field. The main window
will show the gel image, sample list, and chip summary. Any problems detected by the software
will be indicated with yellow triangles above the lanes in the gel image (see troubleshooting).
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1- Ladder:
Select the Ladder sample on the sample list. The main window should show the the following
peaks (11 ladder peaks plus lower-LM- and upper-UM- markers):
15bp Lower Marker
25bp Ladder
50bp Ladder
100bp Ladder
150bp Ladder
200bp Ladder
300bp Ladder
400bp Ladder
500bp Ladder
700bp Ladder
850bp Ladder
1000bp Ladder
1500bp Upper Marker
2—Samples:
Click on the individual sample on the sample list. The positive control and sample lanes should
show two peaks, indicating the HVII and HVI amplified products (around 420-490 bp) for
samples amplified with HVI and HVII multiplex primers. All samples should have the lower
marker (~15bp) and upper marker (~1500bp).
3-Manual editing:
If the upper marker (UM) or lower marker (LM) is present but not labeled properly, right click
on the peak cell “size bp” in the table and select “manually set upper marker” or “manually set
lower marker,” respectively.
If a ladder peak, HVI and/or HVII are present but not labeled or if an extra peak is present: right
click on the peak, select “manual integration”, add or remove peak at that position (bp).
Smaller amplified product peaks in samples with severely unbalanced HVI and HVII peak
heights due to potential length heteroplasmy may be manually edited. Be sure to record any and
all edits within the run documentation.
4-Export data to the network:
The data are automatically saved in a “yyyy-mm-dd” folder as “DES547045xx\yyyy-mm-dd_hhmm-ss.xad” (a shortcut on the desktop). If from AG1: DES547045xx is DES54704515, if from
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AG2:DES547045xx is DES54704524.
a- Open the yyyy-mm-dd_hh-mm-ss.xad file, make edits if necessary, save the .xad file
as DES547045xx\yyyy-mm-dd_hh-mm-ss-analystinitials.xad. Transfer the .xad file (s) in
M:\MITO_DATA\Agilent Archive\yyyy\ yyyy-mm-dd-hh-mm-ss folder.
b- Create a PDF file by going to the “file” menu and selecting “print.” When the print
window opens, select “Run Summary”, “electropherograms” and “Results Table”, choose “all
wells” if it is a full chip, or fill in the well numbers of used wells for a partial chip. Select
“Include Ladder”, one per page, PDF. Click on “… “ to select the drive. Select the appropriate
folder on the network. Add analystinitials before “.pdf” in the path name. Click save.
c- Check that PDF and.xad file (s) are present in M:\MITO_DATA\Agilent
Archive\yyyy\ a new yyyy-mm-dd-hh-mm-ss folder.
2100 expert software
If analysis or review is done at a different time than the run or from another computer, open the
2100 Expert software.
Select “data” in the “contexts” column on the left side of the window.
Go to file
Open
Select the folder with the run you want to review/analyze in M:\MITO_DATA\Agilent
Archive\YEAR\xxxxxxxxxxxxx
Select the appropriate.xad file of the run
Click open
After analysis/review: IF ANY EDITS/CHANGES ARE MADE, do not forget to re-save the
.xad file with -reviewerinitials” at the end of its name. Create a new PDF file. Print and initial.
Section D- Data Entry, Review, Filing, Rerun
DATA TAB ENTRY
Open the appropriate Agilent batch to review and edit, as necessary.
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bp * : peak position manually called
bp ** : peak position edited out manually
bp ^ : spikes position manually removed
LM*: sample lower maker manually called
Samples
HVII and HVI peaks should be discrete and UM*: sample upper maker manually called
approximately 400 to 500bp respectively or the
peak will be inconclusive. Samples with multiple peaks or peak imbalance due to potential
sequence length heteroplasmy may be manually edited.
The “Vol, Used” refers to the volume aliquotted of the original amplified sample.
Sample edits:
If a sample is edited, enter the location in bp followed by one of the symbols from the table
above. If special edits are needed, document them in the sample edits column. Indicate if editing
was necessary for any sample, including the ladder, and if so, record the edits in the
documentation.
Once the editing is complete in the Agilent Software, save the file, and export the final results to
the appropriate network location.
Import this Agilent data file into the LIMS system against the Agilent batch data. This will
automatically update the dilution factors, and input the HVI and HVII initial values. This, in
turn, will calculate the value of HVI and HVII, and output the final “Conc, Mean HVI-HVII”
value for each sample in the batch.
REVIEW TAB ENTRY
Fill the “Intrepretation” column following the guidelines in the table below:
one dilution
HVII
HVI
Comments
[0.5-20]
INC
[0.5-20]
INC
[0.5-20]
[0.5-20]
INC
INC
HVII and HVI mean concentration will be used for further testing
non-INC peak concentration value will be used for further testing
non-INC peak concentration value will be used for further testing
Rerun
2 dilutions
USE
USE
USE
RQ
Comments
dilution A pass dilution A within ± 2.5 X dilution
use lower value
USE
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dilution B pass
B
-
dilution A pass dilution A outside ± 2.5 Xdilution
B
dilution B pass
Rerun
Rerun
RQ
RQ
dilution A pass
dilution B INC
n/a
use for further testing
n/a
USE
-
dilution A INC
dilution B pass
n/a
n/a
use for further testing
USE
If both dilutions are INC
Rerun at appropriate dilution
dilution A INC
RQ, dx
n/a
Rerun at appropriate dilution
dilution B INC
RQ, dx
appropriate dilution example 1
Rerun at appropriate dilution
dilution 2 <0.5
RQ, d1
n/a
Rerun at appropriate dilution
dilution 5 <0.5
RQ, d1
appropriate dilution example 2
Rerun at appropriate dilution
dilution 2 >20
RQ, d10
n/a
Rerun at appropriate dilution
dilution 5 >20
RQ, d100
If both dilutions are INC for any other reason than concentration value out of range, see supervisor
INC = sample inconclusive
USE = concentration will be sued for further testing
RQ = sample will be requantified
dx = sampel will be requantified at dilution x
♦
If the ladder fails (e.g., discrete bands) or upper or lower markers are not present (e.g.,
can’t be edited) the run is inconclusive, all samples have to be requantified, indicate as such in
each sample Interpretation cell.
♦
If one or both markers are not called, the run is inconclusive, all samples have to be
requantified, indicate as such in each sample Interpretation cell.
♦
If a sample peak concentration value is out of range it will appear as INC
If a sample was called INC for any other reason than the value range it will appear as INC. A
comment can be added to explain why the peak was called INC in the “notes” of the review sheet.
In both cases the concentration of only one peak could be used for further testing instead of the
mean concentration of both peaks and USE added in the comments column (see table above).
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If the ladder fails manually select “fail” as the “Ladder Result”, if not select “Pass”.
If the ladder marker (s) fail(s) circle “fail” as the “Ladder Result”, if not select “Pass”.
REVIEW
The reviewer will review the documentation, as well as any comments based on the parameters
in section D.
After review, the reviewer will indicate the appropriate samples for the next process step, as
indicated by the Interpretation results of each sample.
RERUNS
1. After review, the analyst will set up a new Agilent batch for the necessary samples that
were indicated as reruns.
For troubleshooting, refer to the Agilent 2100 Bioanalyzer Maintenance and Troubleshooting
Guide Edition 11/November 2003.pdf archived in MITO_DATA folder.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Mitochondrial DNA Linear Array Analysis
PURPOSE
For the detection of sequence variation targeting 18 positions within the hypervariable regions I
(HVI) and II (HVII) of the human mitochondrial DNA (mtDNA) genome. Large sample sets can
be screened using immobilized oligonucleotide strips via a biotin/streptavidin-linked
hybridization assay (Roche Linear Array Mitochondrial DNA HVI/HVII Region-Sequence
Typing Kit is used), allowing the identification and comparison of questioned samples and
known samples.
PROCEDURE
The following procedure will take approximately 2-2.5 hours. Make sure before beginning the
procedure that sufficient amounts of Linear Array Wash Buffer, Citrate Buffer, and Chromogen
are available. If not, contact the QA team to make or order more. The Linear Array procedure
can accommodate 24 samples per tray, and no more than two trays can be run simultaneously in
one shaking waterbath. If less than 24 samples are being run, leave empty wells between
samples whenever possible to avoid spillover contamination. A single tray should contain only
exemplar samples or evidence samples. Do not run exemplars and evidence together in the
same tray. Do not run evidence or exemplar sample types from the same case in one
Linear Array experiment (e.g. staggering the run of 2 trays in one Linear Array
experiment; one tray contains evidence from a given case, the other tray contains an
exemplar sample from the same case).
A.
Hybridization
1.
Turn on the shaking water bath to 55EC, at 50-70 rpm. Do not leave uncovered.
Be sure that there is enough water covering the base of the bath before proceeding.
2.
Turn on the circulating stationary water bath to 45EC. Warm the Wash Buffer.
3.
Remove samples from storage, vortex and spin down in a centrifuge. Arrange
your amplified samples in a tube rack, and record the Linear Array
documentation.
4.
Using forceps, remove an appropriate number of Linear Array strips; arrange on a
kimwipe, and label the clear end with a pen containing non-soluble ink so that the
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5.
black bar is on the left side of the strip. Place the array strips in the 24-well
tray in the same orientation. Cover the tray with the lid. Take care not to touch
the stripes on the strips with gloves or scrape with forceps.
Label an appropriate number of 1.5 ml microcentrifuge tubes.
6.
Have a witness check the set up.
7.
Add:
- 15 µl of Denaturation Solution
- Up to 21.4 µl of PCR product
Whenever possible, the target amount of 75 ng of amplified mtDNA should be
used for the Linear Array assay.
If the concentration of amplified DNA in an extract is < 10ng / 4 µl (< 2.5ng / µl),
do not use for Linear Array. Reamplify the sample with more input DNA or
proceed directly to DNA sequencing.
8.
Add 3 ml of pre-warmed Wash Buffer to each Linear Array strip.
9.
Add the denatured samples to the liquid within each appropriate well.
Do not add the sample directly onto the strip.
10.
Mix by rocking gently. Cover the tray with the lid, foil, and add two weights on
the top. Place in the shaking water bath for 15 minutes.
When 5 minutes are left on the previous step, prepare the Enzyme Conjugate
solution using the following formula:
Number of samples x 3.3 ml Wash Buffer
Number of samples x 12 µl Enzyme Conjugate
Swirl to mix, cover with foil, and add a weight to the top to prevent from tipping
over. Place in the circulating water bath to keep warm.
11.
Remove the tray from the shaking water bath, uncover, and pour off the Wash
Buffer from the labeled end of the strips. Wipe the condensation from the lid and
edges of the tray.
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12.
Add 3 ml of pre-warmed Wash Buffer to each strip. Rinse by carefully rocking
for 10 seconds and pour off from the labeled end of the strips. Wipe the
condensation from the lid and edges of the tray.
13.
Add 3 ml of the Enzyme Conjugate solution to each strip with a disposable
pipette.
14.
Cover with lid and foil, add weights, and place the tray in the shaking water bath
for 5 minutes.
15.
Remove the tray from the shaking water bath, uncover, and pour off the Enzyme
Conjugate solution from the labeled end of the strips. Wipe the condensation
from the lid and edges of the tray.
16.
Add 3 ml of pre-warmed Wash Buffer to each strip. Rinse by rocking for 10
seconds, and pour off from the labeled end of the strips.
17.
Add 3 ml of pre-warmed Wash Buffer to each strip.
18.
Cover with lid and foil, add weights, and place in the shaking water bath for 12
minutes.
19.
Remove the tray from the shaking water bath, uncover, and pour off the Wash
Buffer from the labeled end of the strips. Wipe the condensation from the lid and
edges of the tray.
20.
Add 3 ml of pre-warmed Wash Buffer to each strip. Rinse by rocking for 10
seconds, and pour off from the labeled end of the strips.
21.
Add 3 ml of Citrate Buffer to each strip.
22.
Cover with lid and foil, add weights and shake at Room Temp for 5 minutes at
100 rpm.
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23.
In a glass container, prepare the Color Development solution using the
following formula:
Number of samples x 3.3 ml Citrate Buffer
Number of samples x 4 µl 3% Hydrogen Peroxide
Number of samples x 0.15 ml Chromogen
Swirl to mix and use immediately.
24.
Remove the tray from the room temperature shaker, uncover, and pour off the
Citrate Buffer from the labeled end of the strips. Wipe the condensation from the
lid and edges of the tray.
25.
Add 3 ml of the Color Development solution to each strip by using a disposable
pipette.
26.
Cover with lid and foil, add weights, and shake at Room Temperature for a
minimum of 10 minutes at 100 rpm. After 10 minutes, briefly check color
development. If strip is not fully developed, continue development for an
additional 5 minutes. Color development is normally complete after 15 minutes;
however, further incubation can be done at the analyst’s discretion. Color
development must be stopped if there is any indication of the appearance of blue
non-specific background staining. Record the total time of color development on
the Linear Array documentation.
27.
Remove the tray from the room temperature shaker, uncover, and pour off the
Color Development solution from the labeled end of the strips. Wipe the
condensation from the lid and edges of the tray.
28.
Stop the color development by adding 5 ml of dH2O to each strip. Rinse by
rocking for 20-30 seconds.
29.
Pour off the dH2O from the labeled ends of the strips. Repeat the dH2O rinse
process twice more. When finished, either immediately proceed to the
photography stage (preferred) or store the strips in a hybridization tray filled with
dH2O (not more than several hours) and photograph later. Record the date, time
and a brief description of samples on the lid.
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30.
B.
Clean trays with ethanol or isopropanol and rinse with dH2O. If after rinsing with
ethanol or isopropanol and the dye is still present, repeat the cleaning, but let
stand for half an hour, and then rinse.
Photography
Using forceps, arrange the developed Linear Array strips on an acrylic tray, using the
supplied ruler from Roche as a guide. The black bar on the left side of the strip should be
used to align the strips to the ruler. Be sure to keep the strips moist.
No more than 12 strips should be photographed at a time. If there are more than 12
strips present, the second photograph needs to include a positive and a
(amplification or extraction) negative control strip taken from the first set of strips.
1. Once arranged on the tray, place the tray onto the copy stand. Turn on spotlights as
necessary.
2. Attach the digital camera to the copy stand by screwing it in place. Adjust the height
and focus of the camera so that all of the strips and ruler are visible and clearly
focused. Optimal settings include setting the camera to the macro mode (which is the
flower image beneath the OK button); manual focusing the image by pressing half
way down on the shutter release button; and by also adjusting the white balance by
selecting Menu: White Balance: White Bal. Preset, select Measure, and then frame
the reference object (with any light sources turned on) and press OK.
3. Connect the camera to the computer, open MS Powerpoint and insert the image into a
blank slide. Using a text box label the image with the picture’s reference number as
LAmmddyy-hhmm from the associated Linear Array worksheet date and time. Save
.ppt file to into M:\MITO_DATA\Photo Archives\Linear Array Photo Archive folder
using the picture’s reference number. Export the image as a .JPEG file, and save into
the same network location. Attach this .JPEG file to the LIMS Linear Array test
batch. Print image, initial and date the picture, and proceed to interpret the results.
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A
B.
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Interpretation of Results
Allele Calling
1.
All interpretations must be made from the photo documentation of the Linear
Array strips.
2.
Fill in the corresponding bands on the Linear Array documentation based on the
ruler markings.
3.
Weak bands are considered any bands that have an intensity significantly
less than the other bands within the same hypervariable region on the same
strip. All weak bands should be recorded as “wX”, where X is the actual probe
signal.
4.
In the case where two bands are present at one probe location, record both bands
as “X/Y” where X is the first band numerically and Y is the second.
The weak band rule may still apply, such that “wX/wY”, “wX/Y” and “X/wY”
are all the possibilities.
5.
If no bands are present at a probe location, enter “0” for that location in the
spreadsheet. If no bands are present in any of the probe locations, all locations
should be marked with a dash “-“.
Control Sample Guidelines
1.
Check the negative controls to make sure there are no bands present.
2.
The HL60 positive control, run with each amplification set, should yield the
following probe signals:
Probe
HL60
3.
C.
16093
1
1A
1
1C
1
1D
2
1E
2
IIA
2
IIB
6
IIC
1
IID 189
1
1
See “INTERPRETATION GUIDELINES - GUIDELINES FOR CONTROLS”
on how to proceed if there are any problems.
Interpretation and further testing strategy
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1.
See “INTERPRETATION GUIDELINES - LINEAR ARRAY” for comparison of
allele calls.
2.
Based on the case context, the following samples need to proceed to cycle
sequence analysis:
- Included exemplars
- One representative sample for all probative evidence
- Samples with partial profiles
- All negative and positive controls if any sample from the batch is being
sequenced
No sequencing is required for:
- Excluded exemplars
- Apparent mixtures
- Redundant evidence samples
IMPORTANT: if any linear array re-hybridization is required, make sure sufficient
amplification product for cycle sequencing is preserved. This is especially valid for
the negative controls and samples with low DNA yields that cannot easily be reamplified.
Depending on the case it might be necessary to omit the linear array repeat.
D.
Sequence concordance
1.
For all single source samples with probe signals, the linear array results are
correlated to the underlying sequence variants for concordance checking.
2.
This is done in an automated fashion using the Linear Array Summary & Stats
spreadsheet found on the network.
3.
On the first page of the spreadsheet, fill in the sample name.
A.
Fill in the probe signals interpreted from the previous section into the
corresponding cells.
The following rules must be applied:
1)
Input weak bands with a “w” followed by the numerical type.
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2)
Input multiple bands, e.g., x/y, on two separate sheets, one with the
“x” and one with the “y,” with the following exception. If a
w2/w3 call is present at 1C, then enter “w2/w3” at this location.
3)
Input null bands with a zero.
B.Print out the spreadsheet to include in the casefile.
C. Print out the results to a pdf document, and attach the pdf to the LIMS for the Linear
Array test batch.
4.
The spreadsheet will provide the following sequence for the HL60 positive
control:
Probe Designation
16093
1A
1C
HVI
1D
1E
IIA
IIB
HVII
IIC
IID
189
Sequence Variation
16093 T
16126 T
16129 G
16304 T
16309 A
16311 T
16362 C
16270 C
16278 T
73 G
146 T
150 T
152 C
189 A
195 T
198 C
200 A
247 G
189 A
4.Assay results can be translated into sequencing results based on the spreadsheet
calculations, and compared against the other samples. Null alleles are not convertible.
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5.For reference, the probe designations and sequence variations are presented below.
HVII
HVI
Probe Designations
Sequence Variation Detected
16093 1
16093 2
A
•
T
•
16093
T
C
T
•
C
•
1A1
1A2
1A3
T
•
•
G
•
•
16126
T
C
•
A
•
•
C
•
•
1C1
1C2
1C3
1C4
1Cw2/w3
A
•
•
•
•
1D1
1D2
C
•
1E1
1E2
1E3
C
•
•
IIA 1
IIA 2
G
•
T
•
73
A
G
T
•
G
•
IIB1
IIB2
IIB3
IIB4
IIB5
IIB6
IIB7
C
•
•
•
•
•
•
C
•
•
•
•
•
•
146
T
C
•
C
•
•
C
C
•
•
•
•
•
•
A
•
•
•
•
•
•
IIC1
IIC2
IIC4
IIC5
G
•
•
•
189
A
•
•
G
C
•
•
•
IID1
IID2
T
•
T
•
247
G
A
16304
T
C
•
•
C
G
•
•
•
•
G
•
A
•
•
A
•
•
•
A
•
•
•
•
16362
T
C
16270
C
•
T
T
•
•
A
•
•
•
C
•
•
•
•
A
•
•
•
•
C
•
A
•
T
•
•
•
•
G
•
•
16309
A
•
•
G
•
T
•
•
G
•
•
•
•
16311
T
•
C
•
C
A
•
•
•
•
C
•
•
•
•
16278
C
T
•
C
•
•
C
•
A
•
•
T
•
•
•
16129
G
•
A
G
•
•
A
•
•
•
C
•
•
•
G
•
•
A
•
•
150
C
•
•
•
T
T
T
T
•
•
•
•
•
•
195
T
C
C
•
T
•
•
T
•
•
•
A
•
•
•
A
•
•
C
•
•
•
•
•
•
198
C
•
T
•
T
•
•
•
152
T
•
C
C
•
C
C
200
A
•
•
G
A
•
•
•
•
•
•
A
•
•
•
A
•
•
T
•
•
•
•
•
•
A
•
•
•
A
•
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
MITOCHONDRIAL DNA LINEAR ARRAY ANALYSIS
DATE EFFECTIVE
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189 1
189 2
G
•
A
•
189
A
G
C
•
A
•
T
•
A
•
C
•
195
C/T*
•
* Both 189 1 and 189 2 probes are degenerate at position 195 with respect to the presence of either a C or T base.
TROUBLESHOOTING
Problem:
No bands on a Linear Array strip where there should be.
Possible causes
Quality of reagents is inappropriate
Solutions
Check expiration dates on all reagents, and
make new if necessary.
Make sure Enzyme Conjugate and Color
Development Solutions were not made more
than 5 minutes prior to use.
Check that all reagents were added at accurate
volumes.
All of the proper reagents were not added
Make sure that Citrate and Wash buffers were
not switched at any steps.
Check quantitation assay.
No DNA present
Check other strips: problem may be isolated
to one well or PCR sample.
Check temperature of water bath, and ensure
that all steps were done at the proper
temperature.
Temperature
DNA was not denatured properly
Make sure Denaturation Solution was added
to sample.
Make or purchase new Denaturation Solution.
DNA degradation
Do not allow DNA to incubate with
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MITOCHONDRIAL DNA LINEAR ARRAY ANALYSIS
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Denaturation Solution for more than 60
minutes.
Problem:
High Background
Possible causes
Color Development step not performed at
room temperature or temperature of the room
is too high.
Insufficient or improper water washes
following Color Development.
Excess Color Development Solution
remaining in tray following aspiration of
pouring-off of solution.
Developed strips exposed to strong light and
/or not kept wet during interpretation or
imaging.
Inadequate agitation of the strips during
Hybridization, Conjugation, and or Wash
steps.
Excess Amount of Enzyme Conjugate: SAPOD added to Enzyme Conjugate Solution.
Tray not properly cleaned prior to use.
Wash bottles are contaminated.
Solutions
Repeat typing and ensure that color
development step is performed on an orbital
shaker at room temperature, between +15 and
+25EC.
Ensure that the strips were washed 3 times for
at least 20-30 seconds per wash: wash times
can be increased or additional washes can be
performed.
Ensure Color Development Solution is
adequately removed.
De-ionized water can be applied with a squirt
bottle during photography if necessary. Align
all strips prior to turning on photography
lights.
Check speed of rotating water bath (50-70
rpm). Verify that solutions are washing over
strips. Repeat typing.
Check calculations and repeat typing,
ensuring correct amount of Enzyme
Conjugate:SA-POD added to Enzyme
Conjugate Solution.
Ensure trays were not cleaned with detergents
or bleach; thoroughly clean and dry the
affected trays. Clean trays with ethanol or
isopropanol and rinse with dH2O. Discard
tray if discolored even after proper cleaning.
Thoroughly clean and dry wash bottles.
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MITOCHONDRIAL DNA LINEAR ARRAY ANALYSIS
DATE EFFECTIVE
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THIS PAGE INTENTIONALLY LEFT BLANK
Revision History:
July 24, 2010 – Initial version of procedure.
September 3, 2010 – Revised version of procedure: Paragraphs B3 and B4 have been rewritten into one paragraph B3 only
to reflect our current procedure
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
EXO-SAP-IT SAMPLE CLEANUP
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EXO-SAP-IT Sample Cleanup
PURPOSE: Prior to cycle sequencing, unincorporated primers and nucleotides present in the
amplification reaction are deactivated by the addition of ExoSAP-IT.
PROCEDURE:
1.
Create a new ExoSAP-It test batch in the LIMS system, and fill in the necessary
documentation.
2.
Confirm the tube label and sample description for each sample. Every run should include
a positive control and an amplification negative control. Note: It is very important for
these entries to be in 3130xl format; do not use spaces or the following characters: \ /
: * “ > < | ? ‘)
3.
Based on each sample’s previous runs, the appropriate values for each column in the
ExoSAP-It batch will be automatically filled in. The “Vol, Misc” must be entered by the
analyst, and then the batch should be saved. This will trigger the automatic values to
populate in the data, and the calculations for the ExoSAP-It volume and the new
concentration will automatically execute.
For a detailed description of the calculations performed in this spreadsheet, refer to
Appendix D – Detailed CycSeq/3130xl Spreadsheet Calculations.
There should be 1ul of ExoSAP-IT added for every 5ul of sample in the
amplification tube.
4.
Use the following settings to incubate the samples:
9700 Thermal Cycler
User: mtDNA
The ExoSAP-IT file is as follows:
- Soak at 37°C for 15 minutes
- Soak at 80°C for 15 minutes
File: exosap-it
Storage soak at 4°C indefinitely
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EXO-SAP-IT SAMPLE CLEANUP
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5.
Place the tubes in the tray in the heat block, slide the heated lid over the tubes, and fasten
the lid by pulling the handle forward.
6.
Start the run by performing the following steps:
a.
The main menu options are RUN CREATE EDIT UTIL USER. To select an
option, press the F key directly under that menu option.
b.
Verify that the user is set to “mtDNA.” If not, select the USER option (F5) to
display the “Select User Name” screen.
c.
Use the circular arrow pad to highlight “mtDNA.” Select the ACCEPT option
(F1).
d.
Select the “exosap-it” file, and press the RUN button (F1).
e.
Verify that the reaction volume is set to 50 :l and the ramp speed is set to 9600
(very important).
f.
If all is correct, select the START option (F1).
The run will start when the heated cover reaches 37oC. The screen will then display a
flow chart of the run conditions. A flashing line indicates the step being performed; the
hold time is counted down. Cycle number is indicated at the top of the screen, counting
up.
Upon completion of the amplification, remove samples and press the STOP button
repeatedly until the “End of Run” screen is displayed. Select the EXIT option (F5).
Wipe any condensation from the heat block with a Kimwipe and pull the lid closed to
prevent dust from collecting on the head block. Turn the instrument off.
7.
When the batch is complete, the new concentration value must be “pushed” to the
ExoSAP-It’d DNA sample within the LIMS system. From this point forward, the
ExoSAP-It’d DNA sample will be the point of all cycle sequencing testing for mtDNA
analysis.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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CYCLE-SEQUENCING
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Cycle-Sequencing
PURPOSE: Following the duplex mtDNA amplification, the samples identified as probative
will be sequenced to determine the mtDNA profile. The Sanger method is used to cycle
sequence the mtDNA in question using fluorescent dideoxynucleoside triphosphate bases chain
terminators. The Applied Biosystems Big Dye Terminator Cycle Sequencing Kit is used.
PROCEDURE:
1.
The cycle sequencing reactions are done in a 96-well plate. Prepare the samples and
reagents needed for cycle sequencing and be witnessed according to the sample names
and order listed on the Cycle Sequencing documentation.
2.
The amount of template DNA and water needed for each sample is calculated by the
LIMS system. This calculation takes into account the total volume and concentration of
amplified product present in the sample tube following the ExoSAP-IT procedure.
The target amount for cycle sequencing is 5 ng of amplified product.
The following formula is then used to create each sample for cycle sequencing
4 :l of Big Dye Terminator Ready Reaction Mix + 2 :l of Sequencing Buffer + 3.2 :l
Primer (1 :M concentration) + mtDNA template + Water = 20 :l total volume.
Samples with less than 5 ng of amplified product in 3:l may be cycle-sequenced using
3:l of the sample.
3.
If a dilution of template DNA is necessary, it will be indicated on the cycle sequencing
documentation as “x @ 1/10th" where x is the input volume. If no dilution is necessary,
the the notation will be “neat.” The amount of water sufficient to make 20Fl reaction
volume is then calculated. If a sample is a negative control sample, the mtDNA
concentration of zero will result in a default template input of 3Fl, with the notation of
“control”attached to the sample The documentation cannot indicate dilution factors
greater than 1/10. For situations where the amount of DNA indicated is less than 1Fl
@1/10 dilution, calculate the volume of extract required @1/100 dilution (multiplication
by 10), and use this volume of a 1/100 dilution. Also calculate, by subtraction, the
correct volume of water to add to the reaction. Note the correct aliquots on the
documentation as a deviation of the procedure.
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CYCLE-SEQUENCING
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For a detailed description of the calculations performed in this spreadsheet, refer to Appendix
D- Detailed Cycle Sequencing/3130xl Spreadsheet Calculations.
4.
A master mix for each primer can be made with the following formula:
a.
For (N+2) samples, add:
• 4 :l x (N+2) Big Dye Terminator Ready Reaction Mix
• 2 :l x (N+2) Sequencing Buffer
• 3.2 :l x (N+2) primer (1uM concentration)
A master mix for each sample DNA can be made with the following formula:
b.
For N samples, add:
• X :l x (N) mtDNA sample DNA, where X is the amount of mtDNA needed as
calculated by the system.
• Y :l x (N) Water, where Y is the amount of water needed as calculated by the
system
Note: The calculations for the two master mixes mentioned above are done by the LIMS
system. They are located by clicking on the “Reagents” tab.
c.
Include all controls for each primer that is used for a sample. If a sample is
repeated starting at the cycle sequencing step the original negative controls do not
have to be repeated if the first test was successful.
d.
The re-cycle sequencing step requires the following:
•
•
•
A new cycle sequencing amplification negative control for each primer
used in re-cycle sequencing to account for the cycle sequencing reagent.
A positive control, for each primer used in re-cycle sequencing to report
on the integrity of the reaction.
Samples can be re-cycle sequenced with more (-recych) or less (-recycl) input
DNA if necessary. Based on validation, up to 90ng of DNA can be used for
recych. If recych sample volume would be more than 3µL, see supervisor.
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CYCLE-SEQUENCING
5.
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Use the following settings to amplify the cycle sequencing samples:
9700 Thermal Cycler
The cycle sequencing amplification file is as follows:
User: mtDNA
Soak at 96EC for 1 minute
25 cycles:
- Denature 96EC for 15 seconds
- Anneal at 50EC for 1 seconds
- Extend at 60EC for 1 minutes
File: BDT cycle seq
Storage soak at 4EC indefinitely
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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SDS CLEANUP
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SDS Cleanup
PURPOSE: To help separate the primers from the cycle-sequenced DNA with the addition of
2% SDS to the samples, prior to Centri-Sep filtration.
PROCEDURE:
Do not refrigerate the 2% SDS tubes. This will cause the SDS to precipitate out of solution.
Store the 2% SDS tubes at room temperature. Ensure that there is no precipitate in the
tube before adding to samples.
1.
Add 2Fl of 2% SDS to each tube of cycle-sequenced DNA. Vortex and spin down the
plate(s) in a centrifuge.
2.
Place the tubes in a thermal cycler, using the following conditions9700 Thermal Cycler
User: mtDNA
The 2% SDS incubation file is as follows:
Soak at 98EC for 5 minutes
Storage soak at 25EC for 10 minutes
File: SDS
3.
When the tubes are back to room temperature following the 25EC soak, proceed to the
Centri-Sep purification.
Revision History:
July 24,2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
CENTRI-SEP SAMPLE FILTRATION
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Centri-Sep Sample Filtration
PURPOSE: Prior to sample electrophoresis, sequenced products must be purified in order to
remove unincorporated dye terminators.
A.
Procedure for Single Columns
1.
Gently tap columns to insure dry gel material has settled to bottom of spin
column. Remove top column cap and add 800 :L of sterile dH2O to one column
for each sequencing reaction.
2.
Replace top cap and mix thoroughly by inverting column and vortexing briefly. It
is important to hydrate all of the dry gel. Allow columns to hydrate for at least 2
hours at room temperature. As the columns are hydrating you will need to label
one sample collection tube (1.5 mL microcentrifuge tube) for each sequencing
reaction. You will also need one wash tube for each hydrated column. These do
not need to be labeled.
3.
Once the columns are hydrated, remove any air bubbles by inverting the column
and sharply tapping the column, allowing the gel to slurry to the opposite end of
the column. Stand the column upright and allow the gel to settle while in a
centrifuge tube rack.
4.
Once the gel is settled, remove first the top column cap, and then remove the
column end stopper from the bottom. Allow excess column fluid to drain into a
wash tube by first gently tapping the column into the wash tube then allowing to
sit for approximately 5 minutes. Remove the column from the wash tube, discard
the liquid and reinsert the column into the wash tube.
5.
Spin the assembly at 700 x g for 2 minutes to remove interstitial fluid. Be sure to
note the orientation of the columns. At this point the columns should be used as
soon as possible for the loading of cycle-sequenced DNA product.
6.
Load entire sequencing reaction volume (20 :L) to the top of the gel. Be careful
to dispense sample directly onto the center of the gel bed without disturbing the
gel surface.
7.
Place column into labeled sample collection tube and spin at 700 x g for 2
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CENTRI-SEP SAMPLE FILTRATION
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minutes maintaining original orientation. The purified sample will collect in the
bottom of the tube.
8.
B.
Discard the column and dry the sample in a vacuum centrifuge (approximately
15-20 minutes). Do not over dry samples.
Procedure for Centri-Sep 8 Strips
1.
Determine how many strips are necessary to filter the amplified samples.
Separate the desired number of strips by cutting the foil between the strips with
scissors.
2.
Open the well outlets on each strip by cutting off the bottom edge with scissors.
Cut at the narrowest part of the bottom of the tube.
3.
Peel off the top foil and arrange the strips evenly on deep-well centrifuge plates.
Spin the plates at 750 rcf for 2 minutes to remove the liquid.
4.
Arrange the newly drained strips on a new 96-well plate. Add the amplified
sample to each column.
5.
Once all of the samples are loaded, place the 96-well plate with the Centri-Sep 8
Strips into the centrifuge, and spin at 750 rcf for 2 minutes.
6.
Confirm that all of the samples passed through the strip into the wells of the 96well plate, and discard the Centri-Sep 8 Strip.
7.
Evaporate the samples in the 96-well plate at 75EC in a thermalcycler with the lid
open.
8.
If the samples are not going to be loaded immediately, they should be stored as
dried pellets at 4°C for no longer then 14 days. When ready, proceed to 3130xl
setup.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
ABI 3130xl SEQUENCING
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ABI 3130xl Sequencing
PURPOSE: The 3130xl 16-capillary array system is used to electrophoretically analyze
samples following cycle sequencing and cleanup. The system uses 96-well plates containing the
samples of interest, and can process 16 separate samples with each injection. Sequence data is
generated at the end of the run for downstream sequencing analysis.
A. Setting up a 3130xl Run
1.
Turn on the computer. Make sure computer is fully booted to the Windows
desktop. To login, the User should be “ocmelims” and the password should be
“passw0rd”. If the instrument is not on, turn it on. The status bar light will
change from solid yellow (indicates instrument is booting) to blinking yellow
(indicates machine is communicating with computer) and then to solid green
(indicates instrument is ready for command).
2.
On the desktop, click on the shortcut for the respective instrument’s data file. The
main path to this data file is:
E:\Applied Biosystems\UDC\data collection\data\ga3130xl\Instrumentname
3.
Once there, create a master file using the following format:
“InstrumentnameYear-Run Number Files” (e.g. Batman08-015 Files) within the
appropriate archive folder (e.g. Batman 2008). Move the 3130xl mtDNA files into
this master file.
4.
Open the 3130xl Data Collection v3.0 software by double clicking on the desktop
Icon or select Start > All Programs > AppliedBiosystems > Data Collection >
Run 3130xl Data Collection v3.0 to display the Service Console..
By default, all applications are off indicated by the red circles. As each
application activates, the red circles (off) change to yellow triangles
(activating), eventually progressing to green squares (on) when they are
fully functional.
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NOTE: This process could take several minutes. The Service Console
must not be closed or it will shut down the application.
Once all applications are running, the Foundation Data Collection window will
be displayed at which time the Service Console window may be minimized.
5.
6.
Check the number of injections on the capillary in the LIMS and in the
Foundation Data Collection window by clicking on the ga3130xl > instrument
name > Instrument Status. If the numbers are not the same, update the LIMS
system. If the number is ≥ 140, notify QC. Proceed only if the number of
injections you are running plus the usage number is ≤ 150.
Check the LIMS to see when the POP6 was last changed. If it is >7 days, proceed
with POP6 change (See part F of this Section) and then return to Step 9.
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7.
Check the level of POP6 in the bottle to ensure there is enough for your run
(approximately 600 µl is needed per injection). If there is not, proceed with POP6
change (See part F of this section) and then return to Step 9.
8.
If you are the first run on the instrument of the day, proceed with steps 9 - 17. If a
run has already been performed on the instrument that day, skip to “Creating a
Plate ID”
9.
Close the instrument doors and press the tray button on the outside of the
instrument to bring the autosampler to the forward position.
10.
Wait until the autosampler has stopped moving and then open the instrument
doors.
11.
Remove the three plastic reservoirs from the sample tray and anode jar from the
base of the lower pump block and dispose of the fluids.
12.
Rinse and fill the “water” and “waste” reservoirs to the line with Gibco® water.
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13.
Make a batch of 1X buffer (45 ml Gibco® water, 5 ml 10X buffer) in a 50mL
conical tube. Record the lot number of the buffer, date of make, and initials on
the side of the tube. Rinse and fill the “buffer” reservoir and anode jar with 1X
buffer to the lines.
14.
Dry the outside and inside rim of the reservoirs/septa and outside of the anode jar
using a Kimwipe and replace the septa strip snugly onto each reservoir. If these
items are not dry, arcing could occur thus ruining the capillary and polymer
blocks.
15.
Place the reservoirs in the instrument in their respective positions, as shown
below:
Water Reservoir
(waste)
Water Reservoir
(rinse)
Cathode Reservoir
(1X buffer)
Empty
2
4
1
16.
Place the anode jar at the base of the lower pump block.
17.
Close the instrument doors
3
B. Creating a Plate ID
1.
Click on the Plate Manager line in the left window.
2.
Select Import from the bottom of the screen. Find the text file that was
previously saved in the master file for the 3130xl run data (e.g. B08-015.txt file
present in the Batman08-015 files folder)
3.
Click on OK.
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C. Preparing the DNA Samples for Sequencing
Arrange amplified samples in a 96-well rack according to how they will be
loaded into the 96- well reaction plate. Sample order is as follows: A1, B1,
C1, D1... G1, H1, A2, B2, C2... G2, H2, A3, B3, C3, etc. Thus the plate is
loaded in a columnar manner where the first injection corresponds to wells A1
to H2, the second injection corresponds to wells A3 to H4 and so on. Label
the side of the reaction plate with the name used for the Plate ID with a
sharpie.
4.
Remove the Hi-Di formamide from the freezer and allow it to thaw. Add 10µl of
formamide to each dried sample and mix to bring the sample into solution.
Once formamide is thawed and aliquoted, discard the tube. Do not re-freeze
opened tubes of Hi-Di formamide.
5.
If single Centri-Sep columns were used, load the entire 10 µl of the resuspended
samples into the 96-well tray in the appropriate wells. The injections are grouped
into 16 wells starting with A1, B1, and so on moving down two columns ending
with 2G, 2H, for a total of 16 wells. Fill any unused wells that are part of an
injection set (eg. containing <16 samples) with 10 µl of Hi-Di formamide.
6.
Once all of the samples have been added to the plate, place the 96-well septa over
the reaction plate and firmly press the septa into place. Spin plate in the
centrifuge for one minute.
7.
Remove the reaction plate from the base and heat denature samples in the 95oC
heatblock for 2 minutes followed by a quick chill in the 4oC chill block for 5
minutes. Centrifuge the tray for one minute after the heat/chill.
8.
Once denatured, place the plate into the plate base. Secure the plate base and
plate with the plate retainer.
IMPORTANT:
Damage to the array tips will occur if the plate retainer
and septa strip holes do not align correctly.
Do not write on the septa with pen, markers, sharpies,
etc. Ink may cause artifacts in samples. Any unnecessary
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markings or debris on the septa may compromise
instrument performance.
D. Placing the Plate onto the Autosampler (Linking and Unlinking Plate)
The Autosampler holds up to two, 96-well plates in tray positions A and
B. To place the plate assembly on the autosampler, there is only one
orientation for the plate, with the notched end of the plate base away
from you.
9.
In the tree pane of the Foundation Data Collection v3.0 software click on GA
Instrument > ga3130xl > instrument name > Run Scheduler > Plate View
10.
Push the tray button on the bottom left of the machine and wait for the
autosampler to move forward and stop at the forward position.
11.
Open the doors and place the tray onto the autosampler in the correct tray
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position, A or B. There is only one orientation for the plate.
12.
Ensure that the plate assembly fits flat in the autosampler. Failure to do so may
allow the capillary tips to lift the plate assembly off the autosampler.
When the plate is correctly positioned, the plate position indicator on the Plate
View page changes from gray to yellow. Close the instrument doors and allow
the autosampler to move back to the home position.
NOTE: When removing a plate from the autosampler, be careful not to hit
the capillary array. Plate B is located directly under the array, so be
especially careful when removing this tray.
Linking/Unlinking the Plate record to Plate
13.
On the plate view screen, click on the plate ID that you are linking. If the plate ID
is not available click Find All, and select the plate ID created for the run.
14.
Click the plate position (A or B) that corresponds to the plate you are linking.
NOTE: It may take a minute for the plate record to link to the plate
depending on the size of the sample sheet.
If two plates are being run, the order in which they are run is based on the order in
which the plates were linked.
Once the plate has been linked, the plate position indicator changes from yellow
to green when linked correctly and the green run button becomes active.
15.
To unlink a plate record just click the plate record you want to unlink and click
“Unlink”.
E. Viewing Run Schedule and Starting Run
1.
In the tree pane of the Foundation Data Collection software, click GA
Instruments > ga3130xl > instrument name > Run Scheduler > Run View.
2.
The RunID column indicates the folder number(s) associated with each injection
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in your run (e.g. Batman-2008-0114-1600-0197). The folder number(s) and the
run ID should be recorded in the LIMS.
3.
Click on the run file to see the Plate Map or grid diagram of your plate on the
right. Check if the blue highlighted boxes correspond to the correct placement of
the samples in the injections.
4.
NOTE: Before starting a run, check for air bubbles in the polymer blocks. If
bubbles are present, click on the Wizards tool box on the top and select
“Bubble Remove Wizard”. Follow the wizard until all bubbles are removed.
5.
Click on the green Run button in the tool bar when you are ready to start the run.
When the Processing Plate dialog box opens (You are about to start processing
plates…), click OK.
6.
To check the progress of a run, click on the Cap/Array Viewer or Capillaries
Viewer in the left window. The Cap/Array Viewer window will show the raw
data of all 16 capillaries at once. The Capillaries Viewer window will show you
the raw data of the capillaries you select to view.
IMPORTANT: Always exit from the Capillary Viewer and Cap/Array
Viewer windows. During a run, do not leave these pages open for extended
periods. This may cause unrecoverable screen update problems. Leave the
Instrument Status window open.
The visible setting should be:
EP voltage 12.2 kV
Laser Power prerun 15 mW
Laser current (no set value)
EP current (no set value)
Laser Power during run 15mW
Oven temperature 50oC
Expected values are: EP current constant around 40-60 µA starting current
EP current constant around 70-80 µA running current
Laser current: 5.0 A + 1.0 A
It is good practice to monitor the initial injections in order to detect problems.
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F. Water Wash and POP Change
Refer to Section A, pg. 2 for schematic of 3130xl while proceeding with the water
wash and POP change procedure.
1.
Remove a new bottle of POP6 from the refrigerator.
2.
Select Wizards > Water Wash Wizard
3.
Click “Close Valve”
4.
Open instrument doors and remove the empty POP bottle.
5.
With a dampened Kimwipe®, wipe the polymer supply tube and cap. Dry.
6.
Replace POP bottle with the water bottle filled to the top with Gibco® Water.
7.
Remove, empty, and replace the anode buffer jar on the lower polymer block.
8.
Click “Water Wash.” This procedure is will take approximately 4 minutes.
9.
When the water wash is finished click “Next”
10.
Select “Same Lot” or “Different Lot”
11.
Remove water bottle from the lower polymer block. Dry supply tube and cap with
a Kimwipe®.
12.
Replace with a new bottle of room temperature POP.
13.
Click “Next.”
14.
Click “Flush.” This will take approximately 2 minutes to complete.
15.
Inspect the pump block, channels, and tubing for air bubbles.
16.
Click “Next.”
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3130xl Genetic Analyzer Troubleshooting
Instrument Startup
Observation
Possible Cause
Recommended Action
No communication between Instrument not started up
the instrument and the
correctly.
computer (yellow light is
blinking).
Make sure the oven door is
closed and locked and the front
doors are closed properly. If
everything is closed properly,
start up in the following
sequence:
a. Log out of the computer.
b. Turn off the instrument.
c. Boot up the computer.
d. After the computer has
booted completely, turn the
instrument on. Wait for the
green status light to come on.
e. Launch Data Collection
software.
Red light is blinking.
Start up in the following
sequence:
a. Log out of the computer.
b. Turn off the instrument.
c. Boot up the computer.
d. After the computer has
booted completely, turn the
instrument on. Wait for the
green status light to come on.
e. Launch the Data Collection
Software.
Incorrect start up procedure.
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Observation
Possible Cause
Recommended Action
Computer screen is frozen.
Communication error. This
may be due to leaving the
user interface in the
Capillary View or Array
View window.
There will be no loss of data.
However, if the instrument is in
the middle of a run, wait for the
run to stop. Then, exit the Data
Collection software and restart as
described above.
Restart the system, and then
press the Tray button.
Autosampler does not move Possible communication
error,
to the forward position.
Communication within the
computer is slow.
OR
OR
Oven or instrument door is
not closed.
a. Close and lock the oven
door.
b. Close the instrument doors.
c. Press the Tray button.
Database is full.
Old files need to be cleaned out
of the database. Follow proper
manual procedures described in
the ABI Prism 3130xl Genetic
Analyzer User’s Manual.
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Spatial Calibration
Observation
Unusual peaks or a flat line
for the spatial calibration.
Possible Cause
Recommended Action
The instrument may need
more time to reach stability.
An unstable instrument can
cause a flat line with no peaks
in the spatial view.
Check or repeat spatial
calibration.
Improper installation of the
detection window.
Reinstall the detection
window and make sure it fits
in the proper position.
Broken capillary resulting in a Check for a broken capillary,
bad polymer fill.
particularly in the detection
window area. If necessary,
replace the capillary array
using the Install Array
Wizard.
Persistently bad spatial
calibration results.
Dirty detection window.
Place a drop of METHANOL
onto the detection window,
and dry. Use only light air
force.
Bad capillary array.
Replace the capillary array,
and then repeat the
calibration. Call Technical
Support if the results do not
improve.
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Spectral Calibration
Observation
No signal.
Possible Cause
Incorrect preparation of
sample.
Recommended Action
Replace samples with fresh
samples prepared with fresh
formamide.
Air bubbles in sample tray.
Centrifuge samples to remove
air bubbles.
If the spectral calibration fails, Clogged capillary
or if a message displays “No
candidate spectral files
found”.
Spike in the data.
Refill the capillaries using
manual control. Look for
clogged capillaries during
capillary fill on the cathode
side.
Incorrect parameter files
and/or run modules selected.
Correct the files and rerun the
calibration.
Insufficient filling of array.
Check for broken capillaries
and refill the capillary array.
Expired matrix standards
Check the expiration date and
storage conditions of the
matrix standards. If
necessary, replace with a fresh
lot.
Expired polymer.
Replace the polymer with
fresh lot using the change
Polymer Wizard.
Air bubbles, especially in the
polymer block tubing.
Refill the capillaries using
manual control.
Possible contaminant or
crystal deposits in the
polymer.
Properly bring the polymer to
room temperature; do not heat
to thaw rapidly. Swirl to
dissolve any solids. Replace
the polymer if it has expired.
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Run Performance
Observation
Possible Cause
No data in all
capillaries
Bubbles in the system.
No signal.
Dead space at bottom of sample
tube.
Low signal strength.
Recommended Action
Visually inspect the polymer
block and the syringes for
bubbles. Remove any bubbles
using the Change Polymer
Wizard. If bubbles still persist,
perform the following:
a. Remove the capillary array.
b. Clean out the polymer bottle.
c. Replace polymer with fresh
polymer.
Centrifuge the sample tray.
Bent capillary array.
Replace the capillary array
Failed reaction.
Repeat reaction.
Cracked or broken capillary
Visually inspect the capillary
array including the detector
window area for signs of
breakage.
Poor quality formamide.
Use a fresh lot of formamide
Insufficient mixing.
Vortex the sample thoroughly,
and then centrifuge the tube to
condense the sample.
Weak amplification of DNA
Re-amplify the DNA.
Instrument/Laser problem
Run instrument diagnostics.
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Observation
Elevated baseline
Possible Cause
Possible contamination in the
polymer path.
Recommended Action
Wash the polymer block with
hot water. Pay particular
attention to the pump block, the
ferrule, the ferrule screw, and
the peek tubing. Dry the parts
by vacuum pump before
replacing them onto the
instrument.
Possible contaminant or crystal
deposits in the polymer.
Bring the polymer to room
temperature, swirl to dissolve
any deposits. Replace polymer
if expired.
Poor spectral calibration.
Perform new spectral
calibration.
Detection cell is dirty.
Place a drop of methanol onto
the detection cell window.
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Observation
Loss of resolution.
Possible Cause
Too much sample injected.
Recommended Action
Dilute the sample and reinject.
Poor quality water.
Use high quality, ultra pure
water.
Poor quality or dilute running
buffer.
Prepare fresh running buffer.
Poor quality or breakdown of
polymer.
Use a fresh lot of polymer.
Capillary array used for more
than 150 injections.
Replace with new capillary
array.
Degraded formamide.
Use fresh formamide and ensure
correct storage conditions.
Improper injection and run
conditions.
Notify QA to check default
settings.
Poor resolution in some Insufficient filling of array.
capillaries.
Refill array and look for
cracked or broken capillaries. If
problem persists contact
Technical Support.
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Observation
No current
Elevated current.
Possible Cause
Poor quality water.
Recommended Action
Use high quality, ultra pure
water.
Water placed in buffer reservoir
position 1.
Replace with fresh running
buffer.
Not enough buffer in anode
reservoir.
Add buffer up to fill line.
Buffer is too dilute.
Prepare new running buffer.
Bubbles present in the polymer
block and/or the capillary and /or
peek tubing.
Pause run and inspect the
instrument for bubbles. They
may be hidden in the peek
tubing.
Open fresh lot of polymer and
store at 4oC.
Decomposed polymer.
Incorrect buffer dilution.
Prepare fresh 1X running
buffer.
Arcing in the gel block.
Check for moisture in and
around the septa, the reservoirs,
the oven, and the autosampler.
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Observation
Fluctuating current
Poor performance of
capillary array used for
fewer than 150 runs.
Migration time
becomes progressively
slower.
Possible Cause
Bubble in polymer block.
Recommended Action
Pause the run, check the
polymer path for bubbles, and
remove them if present.
A slow leak may be present in the
system.
Check polymer blocks for leaks.
Tighten all fittings.
Incorrect buffer concentration.
Prepare fresh running buffer.
Not enough buffer in anode.
Add buffer up to the fill line.
Clogged capillary.
Refill capillary array and check
for clogs.
Arcing.
Check for moisture in and
around the septa, the reservoirs,
the oven, and the autosampler.
Prepare fresh formamide and
reprep samples.
Poor quality formamide
Incorrect buffer.
Prepare new running buffer.
Poor quality sample, possible
cleanup needed.
Desalt samples using a
recommended purification
protocol (e.g., microcon).
Tighten all ferrules, screws and
check valves. Replace any
faulty parts.
Leak in the system.
Improper filling of polymer
block.
Check polymer pump force. If
the force needs to be adjusted,
make a service call.
Expired polymer.
If necessary, change the lot of
polymer.
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Observation
Possible Cause
Recommended Action
Migration time
becomes progressively
faster.
Water in polymer bottle resulting
in diluted polymer.
Replace the polymer, making
sure the bottle is clean and dry.
Arcing in the anode –
lower polymer block.
Moisture on the outside of the
lower polymer block.
Dry the lower block. If
damaged, replace lower
polymer block.
Error message, “Leak
detected” appears. The
run aborts.
Air bubbles in the polymer path.
Check for bubbles and remove
if present, then check for leaks.
Pump block system is
loose/leaking.
Make sure all ferrules, screws,
and tubing is tightly secure.
Ferrule in capillary end of block
may be positioned wrong or
missing. Check for this ferrule.
Lower pump block has burnt out.
When there is condensation in the
reservoir(s) this will cause
electrophoresis problems and
burn the lower block
Air bubbles in the polymer path.
Replace the lower block.
Lower polymer block is not
correctly mounted on the pin
valve.
Check to make sure the metal
fork is in between the pin holder
and not on top or below it.
Buffer jar fills very
quickly with polymer.
Check for bubbles and remove
if present. Then, look for leaks.
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Observation
Possible Cause
Recommended Action
Detection window pops
out while replacing the
capillary array.
Replacing the window
in the correct
orientation is difficult.
Tightening of the array ferrule
knob at the gel block causes high
tension.
Loosen the array ferrule knob to
allow the secure placement of
the window. Re-tighten and
close the detection door.
Detection window
stuck. It is difficult to
remove when changing
the capillary array.
To loosen the detection
window:
a. Undo the array ferrule knob
and pull the polymer block
towards you to first notch.
b. Remove the capillary comb
from the holder in the oven.
c. Hold both sides of the
capillary array around the
detection window area, and
apply gentle pressure equally on
both sides.
d. Release.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Mitochondrial DNA Sequencing Analysis
PURPOSE:
To compile the sequence data generated by the ABI 3130xl into a project for
analysis, by editing the sequence data and compiling a consensus sequence that
can be compared with the revised Cambridge Reference Sequence (rCRS) to
determine the mitochondrial DNA type.
PROCEDURE:
The data following the 3130xl run will be saved on the local 3130xl computer in separate
injection folders. These folders, along with the run statistics and the run sheets will be saved into
a folder in the Mito_Data drive. The run data is also copied into an Analysis Folder in the
Mito_Data drive for analysis. Samples run using the ABI Big Dye Terminator kit will need to be
processed using ABI Sequence Analysis software for the basecalls to be assigned. Once the files
have been processed with Sequence Analysis, they will be imported into the GeneCodes
Sequencher software alignment program for consensus sequence analysis and interpretation of
the mitochondrial DNA type.
A.
Transfer of the 3130xl run data into the master file
1.
On the desktop, click on the shortcut for the respective instrument’s data file. The
main path to this data file is:
E:\Applied Biosystems\UDC\data collection\data\ga3130xl\Instrumentname
Once there, identify the injection folders of the runs you wish to analyze.
2.
Copy these injection folders into the master file (e.g. Batman08-015 files) that
was created earlier making sure that all of the run statistic files and log files are
included.
3.
Also copy the newly created run files into the respective archive files in the MITO
DATA directory.
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Sequence Analysis
1.
Open the Sequence Analysis program by double clicking on the icon. Login
using your username and password.
2.
Click on the import samples icon in the upper left of the screen, or go to Add
Sample(s) under the File menu.
3.
In the new window that pops up, locate the master file of the run that you wish to
analyze. Click on the individual run files within the master file, and for each one
click Add Selected Samples>> at the bottom of the window. As this is done, a
list in the right of the window will populate with the samples from the run. Click
OK when finished importing samples.
4.
The Add Sample Status window will indicate the progress of importing the
samples. When this finishes, the samples will appear in the top window of the
screen. Maximize this area by dragging the center divider bar to the bottom of the
window.
5.
The samples should all have the boxes under BC (base calling) checked. Click on
the Green Arrow at the top of the screen to begin the analysis of the samples.
6.
The Analysis Status window will indicate the progress of analyzing the samples.
As samples are analyzed, the BC column will display a green, blue or yellow box
around the check box indicating the quality of the base calling:
Green:
Indicates a successful base calling for that sample
Blue :
Indicates a problem in base calling the data for that sample
Yellow:
Indicates that there is no data to be analyzed for that sample
7.
If the sequencing analysis is successful, click the yellow floppy-disk icon in the
top left of the screen to Save All Samples, or click on Save All Samples under
the File menu.
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8.
Print the analysis report page. Click on the Analysis Report button at the top of
the page (white button with AR; A is in red and R in blue), or select Analysis
Report under the Analysis menu at the top of the screen. When the report page
opens, right click on the column headings for the analysis report. Deselect the
following options- PP Status, Peak 1, Base Spacing, # Low QV, # Med QV, #
High QV, Sample Score, CR Start, and CR Stop. The visible column headings
will show the following- BC Status, Well, Cap #, LOR, ‘A’ S/N, ‘C’ S/N, ‘G’
S/N, ‘T’ S/N, Avg S/N. To ensure that all sample identification fits onto one row,
deselect “Fit Columns to Window” (lower left of screen), select size “8” font, and
increase the width of “Sample File Name” column.
9.
Create a PDF file of the Analysis Report by clicking Print and send the file to
Adobe PDF. Be sure the page set-up is set to portrait before creating the PDF
file. Check the PDF file to make sure that the complete Sample Names and
Sample Descriptions are present (e.g., not cut off). If necessary, make formatting
adjustments, resend Adobe PDF file, and recheck. Following the successful
creation of the PDF file, save it in the run folder (e.g., save in B08-040 with run
name B08-040 Analysis Report) that is contained within the Analyzed Archive.
10.
Attach the PDF Analysis Report to the LIMS for the cycle-sequencing batch.
Close the PDF Analysis Report and the Analysis Report screen.
11.
Click Exit under the File menu to close out the Sequencing Analysis program.
12.
The Analysis Report will be placed together with the run review sheet, the
3130xl run sheet, and the 3130xl cycle-sequencing worksheet for review and
archiving.
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Sequencher
Before you begin Sequencher, make sure that the USB key is fully inserted in a local
USB port on the computer, and the computer fully recognizes the key. If successful,
the computer will make a “Ding-Dong” sound. Starting Sequencher without this
USB key will cause the program to lock out all editing capabilities.
1.
Open Sequencher. Under the Contig menu, select Consensus to Forensic Standards.
This only needs to be done after opening Sequencher for the first time. Once set, it will
remain until the program is closed. Also, check to make sure that the Assembly
parameters used to create contigs are set to the proper settings. Click on the box in the
upper left-hand corner that is marked “Assembly Parameters”. The following settings
should be selected:
Assembly Algorithm:
Optimize Gap Placement:
Minimum Match Percentage:
Minimum Overlap:
Assemble By Name:
Dirty Data (radial button)
Use ReAligner (check box)
Prefer 3' Gap Placement (check box)
85% (slide bar)
20 (slide bar)
not Enabled (deselected)
2.
Under the File menu, go to Import and select Sequences.
3.
Find the sequence files that were copied to the Analyzed Archive. To simplify,
under File of Type select “With Chromatogram Sequences.” To select all of the
files press and hold the Shift key, and click on the last file. Once the files are
selected, click Open. Maximize the analysis window to view all of the samples
and sample data.
4.
Import the appropriate reference sequence into the project for every contig that
needs to be built. The reference files (HVI.spf or HVII.spf) are located on the
Forensic Biology network (Mito Data/Reference Seqs).
These files can be saved to the desktop or local hard drive for easier access.
5.
Holding down the Shift or the Control key, click on imported rCRS file, and the
forward and reverse sequence files that will make up the contig.
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6.
At the top of the screen, click on the Assemble to Reference button. If the
samples contain viable data, the contig will be formed. If the samples do not
contain quality data, they will not import into the contig. Name the contig
according to the sample name and hypervariable region sequenced. (e.g.- Hair 1A
HVII)
7.
Select the new Contig icon by clicking on it once. In the Contig menu at the top
of the screen, select Trim to Reference Sequence.
8.
Double click on the Contig icon. When the contig diagram window opens, click
on the Bases button at the top left of the screen.
A new window will open showing the individual sequence files above the
rCRS reference sequence at the top of the window and the consensus
sequence at the bottom of the window. Individual sequences, including the
reference sequence can be moved up or down by placing the cursor on the
name of the sequence in the upper left box and dragging the sequence up or
down.
Under the consensus sequence is a series of “•” and “+” symbols. The “•”
symbols highlight base call disagreements from the rCRS and the “+”
symbols highlight ambiguities in the consensus sequence.
9.
To view the chromatogram data and the sequence data together, highlight a base
in the consensus sequence and click the Show Chromatograms button at the top
of the screen. This will open a second window showing the chromatogram data
for all of the sequences in the contig. Notice that the reverse primer sequence has
been reversed and compiled in the process of building the contig. Adjust the
position of the two screens so that all of the sequence data is visible along with
the chromatogram data, and so all of the base positions can be reviewed. Use this
display and review all sequence positions.
10.
To quickly move from one ambiguity to the next in the consensus sequence, click
on the first base in the sequence and then press Control-N simultaneously on the
keyboard. This will jump both the sequence data and chromatograms to the next
ambiguous position. To find only the instances where the contig is in
disagreement between the strand data, click on the sequence data and press
Control-D.
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IMPORTANT- In the instances where length and/or sequence heteroplasmy
is present and yields single-stranded DNA regions and/or ambiguous bases,
or un-editable N calls are present, the consensus setting on the Sequencher
program must be set to Plurality mode to analyze that particular sample.
11.
Within the LIMS, create the appropriate contig samples, and indicate which
primer samples are associated with each appropriate contig. All edits, trims, and
modifications to the contig sequence must be recorded within the LIMS
documentation.
12.
To edit a base call, click on the base in question in the consensus sequence or
individual sequence, and press the appropriate letter on the keyboard according to
the following:
Standard Codes
A- Adenine
C- Cytosine
T- Thymine
G- Guanine
N- Ambiguous
IUPAC Codes *
R- A or G
Y- C or T
K- G or T
M- A or C
S- C or G
W- A or T
*See Nomenclature section of this manual for further discussion.
13.
To delete a base, click on the base in question and do one of the following:
a.
b.
To have the bases fill in from the left side of the strand, press the delete
key.
To have the bases fill in from the right side of the strand, press the
backspace key, and follow the on-screen instructions.
14.
To insert a base, press the Tab key and follow the on-screen instructions.
15.
To shift the entire strand, place the cursor over the strand in question and press
and hold the Ctrl key. The cursor will turn into a open hand icon. Click and hold
using the icon and the hand will “grab” the strand, allowing you to move the
entire strand left or right.
16.
To move a single base, place the cursor over the base in question and press and
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hold the Alt key. The cursor will turn into a lasso icon. Click and hold using the
icon and the lasso will “grab” the base, allowing you to move the base.
17.
To highlight a section of the sequence, click on the beginning base in the
sequence you wish to highlight, and then move the cursor to the last base in the
sequence section, press and hold the Shift key, and click on the final base.
18.
To review edits press and hold Ctrl E.
19.
When the sequencing analysis and editing are completed, the contigs that were
built need to be archived in the appropriate folders. To archive contigs, hightlight
contigs that are to be archived together, go to file export selection as
subproject. Click on Browse button and select folder for contig to be archived.
Make sure that the format is set to ‘Sequencher Project’. Click the Export button.
Enter project name, e.g. PCddmmyy-hhmm for positive control contigs and
FBXX-12345 for sample contigs.
Positive Control contigs should be archived as follows: M:/MITO_DATA/PC
Archive/year/PCmmddyy-hhmm.
Samples contigs should be archived as follows: M:/MITO_DATA/Project
Archives/sample type/FBXX-12345. Sample type should be ‘Casework’ for
evidence and associated exemplars and should be ‘Missing Person cases’ for all
Missing person samples.
•
•
From this point forward, the saved contigs can be further modified with
additional primer runs, edited, and resaved as outlined above.
The following procedures should only be done once the analysis of the contig
is complete and the contig is ready for review.
20.
Once contigs have been exported and archived, open appropriate project, go to the
Contig menu and click on Compare Consensus to Reference. If necessary,
widen the contig name column to view the entire contig name. To print, select
Reports, Entire Table, Open Report, and Print.
Select portrait as the
orientation, and print.
21.
Print the Compare Consensus to Reference page again, but this time send the
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file to Adobe PDF. Click Print. Save the PDF file in the master run file, named
as the sample name eg. “FB05-0005m hair 1 HVI” Close the Adobe window.
Close the Difference Review window. Attach this PDF to the LIMS system
within the contig sample.
22.
At the top of the sequence comparison page, click on the Summary button.
When the Summary View window opens, click on File, Print Setup, and select
“landscape.” Then click on the Ruler button at the top of the page and adjust the
margins (triangles on ruler) and adjust column spacing as needed to print entire
sample ID.
23.
Print the Summary page. To do this, select the File menu and select Print.
24.
Print the Summary page again, but this time send the file to Adobe PDF. Click
Print. Save the PDF file in the master run file, named as the sample name plus
“sum,” eg. “FB05-0005m hair 1 HVI sum.” Close the Adobe window. Attach
this PDF to the LIMS within the contig sample.
25.
Click on the Overview button. In the Contig Diagram, double click on the
individual sequence files. The sequence window of the file will open. Click on
the Show Chromatogram button at the top of the screen. A new window will
open. In the upper left corner is a slider bar, the four-color bases and two buttons:
a dot and a vertical bar. DO NOT CLICK THE VERTICAL BAR, AS THIS
WILL ERASE THE HEADER FROM THE PRINTED PAGE.
Print this chromatogram as edited data, trimmed to the hypervariable region of
interest. To do this, select the File menu and select Print Setup. Select
landscape as the orientation, and print.
a.
b.
If you are printing the forward strand, under Page Range select the
FIRST four (4) pages of the chromatogram.
If you are printing the reverse strand, under Page Range select the LAST
four (4) pages of the chromatogram.
Repeat step 25 for every separate chromatogram file.
26.
Create an Adobe PDF file for this chromatogram as edited data, trimmed to the
hypervariable region of interest. To do this, select the File menu and select Print
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Setup. Select landscape as the orientation, and print to Adobe DPF from the
drop-down list. Click OK. Under the File menu, select Print.
a.
If you are printing the forward strand, under Page Range select the
FIRST four (4) pages of the chromatogram.
b.
If you are printing the reverse strand, under Page Range select the LAST
four (4) pages of the chromatogram.
Confirm that the Adobe PDF is selected, and click Print. Save the PDF file in
the master run file, named as the sample name plus the primer, e.g. “FB05-0005m
hair 1 HVI A4.” Close the Adobe window. Attach this PDF to the LIMS within
the contig sample.
Repeat step 26 for every separate chromatogram file.
27.
Click on the Overview button.
28.
The lines at the top of the diagram indicate the forward and reverse strands in
relation to the rCRS sequence, and the green bar below the lines indicates which
areas of the rCRS are covered by the available sequence data. If the sequence is
present in both the forward and reverse strand, the bar will be green with thin
white strips on the top and bottom. If there is a partial coverage in only one
direction, the bar will contain a light blue pattern.
29.
Print out the Contig Diagram. Under the File menu, select Print Setup. Select
landscape as the orientation, and print (select printer from the drop-down list).
Click OK. Under the File menu, select Print. Confirm that the appropriate
printer is selected and click Print.
30.
Print the Contig Diagram page again, but this time send the file to Adobe PDF.
Select landscape as the orientation, and click Print. Save the PDF file in the
master run file, named as the sample name plus “map,” eg. “FB05-0005m hair 1
HVI map.” Close the Adobe window. Attach this PDF to the LIMS within the
contig sample.
31.
The final step of Sequencher analysis is to prepare the export data file from
Sequencher for the LIMS. This will allow the contig data to import into the
contig sample within the LIMS. To do this, open the contig and select “Compare
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Consensus to Reference” from the Contig menu. Click on the “Reports” button at
the top of the screen. Select the radio button for “Entire Table” and from the drop
down menu, select the report format as “Variance Table Report.” Save the
variance table as a text file to the appropriate network location and import into the
LIMS for the appropriate contig.
32.
D.
After the import is completed in the LIMS, and the contig data is saved, the
analyst can run the reports for the mtDNA Run Review and Positive Control
Reviews, and print these reports as necessary for review.
File Output and Construction
Arrange the paperwork in following order, from bottom to top:
a.
b.
c.
d.
e.
f.
g.
Run/rerun review sheets
Control review sheets
Sequencher Chromatogram printouts (if necessary)- landscape
Contig Diagram- landscape
Summary View- landscape
Editing Sheet
Compare Consensus to Reference- portrait
Landscape pages should be arranged in the file so that the right side of the landscape
view faces the top edge of the file, and the left side of the landscape view faces the
bottom side of the file.
E.
Data Review
1.
Once all of the Sequencher data has been completed and the file have been
archived, pass the entire set of sample printouts for one 3130 run to another IA for
data review.
2.
For the IA performing the review, the following steps must be performed.
a.
Open the respective file from the appropriate archive located in the
MITO_DATA directory. Review all sequencher files.
b.
For each sample, including Positive Control(s), ensure that all edits
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reflected in the editing documentation are valid and accounted for within
the data set.
3.
F.
c.
For positive controls, ensure that the proper type is displayed on the
Compare Consensus to Reference page.
d.
For negative controls, open the bases and chromatogram windows for each
primer for each sample to ensure that no base calling data is present. If
necessary, attempt to re-build a contig using questionable negative
controls.
e.
Ensure for every sample that the documentation is assembled in order and
reflects the entire project and sample names.
If a problem is found, mark the occurrence within the documentation and return
the documentation to the original analyst.
Archiving the Sequencher Data
Data will be archived in BINDERS, CASEFILES, and ELECTRONIC FILES.
ARCHIVED IN BINDERS:
Instrument binders (e.g. Batman)
3130xl report, original
Analysis report, original
Editing reports for Neg controls that could build into a contig, original
Run review reports, original
PC Binder
For each positive control:
Positive control review report
Contig diagrams, original
Sequence summaries, original
Editing reports, original
Difference review sheets, original
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FB CASEFILES:
3130xl report, copy
Run review report(s), copy
Positive control review report, copy
Positive control editing report, copy
FB sample contigs, originals
Contig diagrams
Sequence summaries
Editing reports
Difference review sheets
ARCHIVED IN ELECTRONIC FILES:
Analyzed run files (e.g. B08-015)
Run files
3130 report
Run review reports
Analyzed run sequence files
Neg controls that could build into a contig
PC archives (e.g. PC_053108-1306)
PC Sequencher file
PC editing report
PC review report
FB project archive (e.g. FB08-12345)
FB Sequencher file
FB editing report
NOTE: If a positive CTR is sequenced twice (e.g., during QC tests), the
name will stay the same as in PCmmddyy-hhmm but a lettered suffix will be
added after each new sequencing as in PC-mmddyy-hhmm-A, PC-mmddyyhhmm-B….
A backup of all of the sequencing data contained in the MITO_DATA directory will be archived
by DOITT.
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Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Sequence Nomenclature and Alignment
Nucleotide positions are designated according to the standard one-letter code based on the
nomenclature system adopted by the International Union of Pure and Applied Chemistry
(IUPAC; see table below). Note that an “N” is used to denote unresolved sequence ambiguities
where N can be any one of the four bases. IUPAC codes that designate two possible bases
should only be used in instances of sequence heteroplasmy.
IUPAC code
Base designation
IUPAC code
Base designation
G
Guanine
R
A or G
A
Adenine
Y
C or T
T
Thymine
K
G or T
C
Cytosine
M
A or C
N
G, A, T, or C
S
C or G
W
A or T
A.Using Sequencher 4.9
1.
Sequence differences between the questioned sample and the revised Cambridge
Reference Sequence (rCRS) are generated and printed out from the Comparison
Report file in Sequencher. These differences are organized by hypervariable
region (eg., one difference review file is generated for each HVI and HVII
region). The differences are listed in order of occurrence on the mtDNA
molecule.
2.
In most cases, the alignment of a given mtDNA sequence with that of rCRS is
straightforward. However, care must be taken in the placement of insertions and
deletions in reference to that of rCRS according to the following standard
nomenclature:
a.
Characterize profiles using the least number of differences from the
reference sequence. Align the 310 T base in the rCRS with a T whenever
possible.
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b.
If there is more than one way to maintain the same number of differences
with respect to the reference sequence, differences should be prioritized in
the following order: (i) substitutions: transitions are favored over
transversions, (ii) insertions/deletions (indels).
c.
Insertions and deletions should be placed furthermost 3' to a
homopolymeric region, with respect to the light strand of rCRS.
Insertions and deletions should be combined in situations where the same
number of differences to the reference sequence is maintained. In
situations involving the “AC” motif, treat this motif as a homopolymeric
region with respect to indels in the AC repeat region. Alignment rules a,
b, and c are described in Budowle, et al, 2007. For casework samples
where alternative alignment following the hierarchy of Wilson, et al,
2002a,b, is also possible, the alternative alignment does not need to be
included in the case file.
3.
Insertions (INS) should be listed to the right of a particular nucleotide position.
Insertions are documented by first noting the site immediately 5' to the insertion
followed by a point and a “1” for the first insertion, a “2” if there is a second
insertion, and so on.
4.
Deletions (DEL) should be listed exactly where the known base in the reference
sequence is missing in the sample sequence to minimize the number of
differences between the questioned sample and the rCRS reference sequence.
Deletions are noted by a “:” on the Sequencher printout in the consensus
sequence.
5.
Sequence heteroplasmy (also known as point or site heteroplasmy) occurs when
a single sample contains at least two mtDNA sequences that differ at one or two
nucleotide positions. The appropriate one-letter IUPAC code will be used during
the editing of a given site that shows sequence heteroplasmy. This designation
will be reflected in the Sequencher Comparison Report. In addition, the presence
of sequence heteroplasmy at the given nucleotide position for the respective
heteroplasmic bases will be recorded on the sequence editing documentation.
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6.
Length heteroplasmy occurs in regions that contain many tandem C nucleotides.
These regions are commonly referred to as polycytosine or C-stretch regions.
Length heteroplasmy refers to a sample that has at least two types, each one
differing by the total number of C nucleotides at a given C-stretch.
a.
It will be noted if a given casework sample has length heteroplasmy in
HVI. The number of C residues, however, in the area with HVI length
heteroplasmy will not be recorded. Length heteroplasmy in HVI most
commonly arises when there is a substitution of a C for a T at position
16,189. The reference type in HVI is C5TC4. Sequences showing length
heteroplasmy in HVI will be truncated to fit the C5TC4 format including
the T to C change at position 16,189.
b.
It will be noted if a given casework sample has length heteroplasmy in
HVII. Length variants in HVII are commonly observed in the number of
C residues preceding a T residue at position 310. It is often possible to
determine unambiguously the dominant length variant in this region. The
profile used for further analysis in Sequencher should be composed of
only the major type as determined by the analyst.
B.Using Sequencher 4.1.4
1.
Sequence differences between the questioned sample and the revised Cambridge
Reference Sequence (rCRS) are generated and printed out from the Difference
Review file in Sequencher. These differences are organized by hypervariable
region (eg., one difference review file is generated for each HVI and HVII
region). The differences are listed in order of occurrence on the mtDNA
molecule. “Ref” (reference) and “Con” (consensus) indicate what bases are
present in the rCRS and the questioned sample, respectively, at the designated
mtDNA sequence positions.
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2.
In most cases, the alignment of a given mtDNA sequence with that of rCRS is
straightforward. However, care must be taken in the placement of insertions and
deletions in reference to that of rCRS according to the following standard
nomenclature:
a.
Characterize profiles using the least number of differences from the
reference sequence. Align the 310 T base in the rCRS with a T whenever
possible.
b.
If there is more than one way to maintain the same number of differences
with respect to the reference sequence, differences should be prioritized in
the following order: (i) substitutions: transitions are favored over
transversions, (ii) insertions/deletions (indels).
c.
Insertions and deletions should be placed furthermost 3' to a
homopolymeric region, with respect to the light strand of rCRS.
Insertions and deletions should be combined in situations where the same
number of differences to the reference sequence is maintained. In
situations involving the “AC” motif, treat this motif as a homopolymeric
region with respect to indels in the AC repeat region. Alignment rules a,
b, and c are described in Budowle, et al, 2007. For casework samples
where alternative alignment following the hierarchy of Wilson, et al,
2002a,b, is also possible, the alternative alignment does not need to be
included in the case file.
3.
Insertions (INS) should be listed to the right of a particular nucleotide position.
Insertions are documented by first noting the site immediately 5' to the insertion
followed by a point and a “1” for the first insertion, a “2” if there is a second
insertion, and so on.
4.
Deletions (DEL) should be listed exactly where the known base in the reference
sequence is missing in the sample sequence to minimize the number of
differences between the questioned sample and the rCRS reference sequence.
Deletions are noted by a “:” on the Sequencher printout in the consensus
sequence.
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5.
Sequence heteroplasmy (also known as point or site heteroplasmy) occurs when
a single sample contains at least two mtDNA sequences that differ at one or two
nucleotide positions. The appropriate one-letter IUPAC code will be used during
the editing of a given site that shows sequence heteroplasmy. This designation
will be reflected in the Difference Review. In addition, the presence of sequence
heteroplasmy at the given nucleotide position for the respective heteroplasmic
bases will be documented on the editing sheet.
6.
Length heteroplasmy occurs in regions that contain many tandem C nucleotides.
These regions are commonly referred to as polycytosine or C-stretch regions.
Length heteroplasmy refers to a sample that has at least two types, each one
differing by the total number of C nucleotides at a given C-stretch.
a.
It will be noted if a given casework sample has length heteroplasmy in
HVI. The number of C residues, however, in the area with HVI length
heteroplasmy will not be recorded. Length heteroplasmy in HVI most
commonly arises when there is a substitution of a C for a T at position
16,189. The reference type in HVI is C5TC4. Sequences showing length
heteroplasmy in HVI will be truncated to fit the C5TC4 format including
the T to C change at position 16,189.
b.
It will be noted if a given casework sample has length heteroplasmy in
HVII. Length variants in HVII are commonly observed in the number of
C residues preceding a T residue at position 310. It is often possible to
determine unambiguously the dominant length variant in this region. The
profile used for further analysis in Sequencher should be composed of
only the major type as determined by the analyst.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Editing Guidelines
Sequencher base calls can be modified if the underlying data support it. The analyst can change
an “N” call into a base determination, insert an additional base, remove a position, or trim a
sequence. A base call must not be edited without proper justification.
Reasons for base removal are:
- Extra base inserted due to broad peak, peak artifact, or analysis default spacing
Reasons for base insertion are:
- Base omitted however authentic peak is present
- To maintain proper spacing
Reasons for changing a base to an “N” or to a degenerate IUPAC code are:
- Ambiguous bases are detected
- Dye or electrophoretic artifact interference
- Due to sequence or length heteroplasmy
Reasons for changing an “N” call to a base is:
- Base omitted or called “N”, however authentic peak is present
- Dye artifact or electrophoretic interference
- Neighboring peak interference
Reasons for trimming a sequence:
- Trimmed to remove end sequence (sequence tail removal)
- Trimmed rCRS and sequences to other (shorter) sequence position for duplication
Editing for other reasons should be documented with a comment explaining the edit.
Many software calls can be easily resolved and corrected by the analyst. However, ambiguous
situations should not be edited. If an electrophoresis problem is suspected, this sample should be
re-injected. Sequence information at each base position should be confirmed by data from both
DNA strands when possible. Single-stranded regions present due to length heteroplasmy, must
be confirmed by confirmatory sequencing of the same strand in the same direction. The
Sequencher complementary strand alignment will flag conflicts between the two sequencing
directions for all strands imported into the contig.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS
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Interpretation Guidelines
GUIDELINES FOR CONTROLS
A.
Negative controls
Negative controls are considered negative if there is no detectable DNA based on the
quantitation procedure and no “readable” sequence is seen after 3130xl electrophoresis.
For DNA sequencing analysis, the controls are also considered negative if sequence was
obtained, but it cannot align to the reference sequence.
A “readable” sequence from a negative control run is a sequence that can be aligned to
the rCRS for >90 consecutive bases with no more than 4 “N” calls within any 10
consecutive bases.
Two negative controls are associated with each sample: the extraction negative (ext neg
or e neg) and the amplification negative (amp neg) controls. The former tests for potential
DNA introduced during extraction through amplification, while the latter tests for the
presence of any background DNA that was introduced during the amplification, or
present in the amplification reagents. Both of these controls need to be processed for all
sequencing primer sets.
Flow charts for passing, failing or retesting negative controls is as follows:
1.
Agilent
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* If confirmed, the following actions should be taken:
•
For extraction negative controls
o Re-amplify to confirm presence of DNA, samples can proceed
if re-amplification is clean.
o If the extraction negative control still yields a peak following
re-amplification, it is preferable to re-extract if more sample is
available. If sample amount is limiting, analyst may proceed
with caution. The results are only valid if the sequence
detected for the amplification negative control does not match
any of the associated samples or any of the samples in the case.
o If the amount of DNA present in the extraction negative
control exceeds 10% of any associated sample (DNA amounts
determined by Agilent), that sample is invalid.
•
For amplification negative controls
o Re-amplify sample set.
o If sample amount is limiting, it is left to the analyst’s discretion
to proceed with the amplification set since this result indicates
that the background DNA is limited to the amplification
control tube rather than being ubiquitous in all samples. The
results are only valid if the sequence detected for the
amplification negative control does not match any of the
associated samples or any of the samples in the case.
o If the amount of DNA present in the amplification negative
control exceeds 10% of any associated sample, that sample is
invalid.
Note: Any failed negative controls may be sequenced for quality control
purposes.
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2.
Linear array
* If confirmed, the following actions should be taken:
•
For extraction negative controls
o Re-amplify and re-hybridize to confirm presence of DNA.
Samples can be interpreted and sequenced if the reamplification does not yield linear array signals.
o If amount of extract available is limited, proceed to sequencing
and do not interpret linear array
•
For amplification negative controls
o Re-amplify sample set.
o If sample amount is limiting, it is left to the analyst’s discretion
to use the data of this amplification set since this result
indicates that the background DNA is limited to the
amplification control tube rather than being ubiquitous in all
samples. The results are only valid if the linear array type
detected for the amplification negative does not match any of
the associated samples or any of the samples in the case.
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3.
Sequencing results
^ If sequence data is present for an extraction or amplification negative control,
but does not have base calls assigned, an analyst should manually assign base
calls to determine if the sequence data can be aligned to the rCRS and if it is a
“readable” sequence.
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* If an extraction or amplification negative control contains a “readable”
sequence, the results should be confirmed by recycle-sequencing. If
confirmed, then the test fails and retesting must start at the point of sample reextraction or amplification. If the amount of original sample present is
limiting, the DNA extract is limiting or the re-amplification yields the same
results, then sample results can be interpreted and reported if the sequence is
different from all associated samples in the case. The determined sequence for
the extraction or amplification negative control must contain “readable”
sequence in order to be used in sequence comparisons with case samples.
If both extraction and amplification negative control from the same test
contains “readable” sequence, the extraction negative cannot be interpreted
because the amplification may have introduced a contaminant. The test fails
and all samples and the extraction negative must be re-amplified and resequenced
**If an extraction or amplification negative controls contain sequence data that
can be aligned to the rCRS for <90 consecutive bases, the test passes however
the results should be confirmed by recycle-sequencing. If confirmed, see a
mtDNA supervisor before proceeding with further testing. The following
testing can be done if further testing is deemed necessary:
• For an extraction negative control, re-amplification of the negative control
in question followed by re-extraction of associated samples if necessary.
• For an amplification negative control, re-amplification of the entire
amplification set.
NOTE: If it is necessary to re-sequence a casework sample from the cycle
sequence step, a new cycle sequencing amplification negative
control (CAN) must be created for this round of cycle sequencing.
This negative control must yield a negative result for the results to
be valid.
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Positive controls
The positive control (HL60) is included for each amplification and must produce
sequence that is consistent with the known polymorphisms. The positive control sample
must yield results for the full read length of the associated sample contig, but at a
minimum, HVI, 16024-16365, HVII, 73-340, or both. In addition, the positive control
serves as the run control. Therefore, in order to be valid, every run must have a positive
control that passes specification.
The known polymorphisms in comparison to the rCRS are as follows:
HVI
16,069
16,193
16,278
16,362
T
T
T
C
HVII
73
150
152
263
295
315.1
G
T
C
G
T
C
This sequence will produce the following linear array type:
Probe
HL60
16093
1
1A
1
1C
1
1D
2
1E
2
IIA
2
IIB
6
IIC
1
IID 189
1
1
If the positive control fails to produce the expected result, all samples associated with this
control fail. If it is suspected that the problem is not related to the amplification but could
stem from a subsequent step, the positive control and all of the samples can be retested
starting either at the cycle sequencing or the 3130xl injection step.
In cases of dye interference or electrophoretic artifact, some N calls in the positive
control will be allowed as follows:
1.
A maximum number of 4 “N” calls within any 10 base stretch for any primer
strand used to build the contig will be allowed for either HVI or HVII region
provided that the calls on the complementary strand are unambiguous and not
contradictory to the questioned nucleotide position(s)
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2.
Any positions that have ambiguous N calls on both complementary strands that
cannot be resolved through retesting will result in the failing of the positive
control and all of the associated sample runs.
Guidelines for Reporting
A.
Linear array interpretation
1.
2.
A sample will not be used for comparisons if:
•
The sample displays a suspected partial profile (generally faint signals, more
than 3 weak probe signals and/or the presence of 4 or more blank regions
indicate the presence of a partial profile)
•
The sample consists of a mixture of DNA (more than 2 apparent
heteroplasmic types could be caused by a mixture)
The numeric probe signal calls are the basis for linear array interpretation.
Conclusions are as follows:
Consistent (cannot
exclude)
All numerical calls match and no locus is inconclusive
Inconclusive
The two types show one difference or one or more of the
regions are inconclusive (see below)
Exclusion
Two or more differences that show no evidence of
heteroplasmy
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3.
Heteroplasmy, weak probe signals or type 0 signals are treated as follows:
Weak probe
signal
Type 0 probe
signal
Heteroplasmy
B.
Weak call versus normal call;
different probe signal
Difference
Weak call versus normal call; same
probe signal
Inconclusive
Weak call versus type 0 probe signal
Inconclusive
Type 0 signal call versus normal call
Difference
Type 0 signal call versus weak probe
Inconclusive
signal
Type 0 signal call versus type 0
signal call
Consistent if no
evidence of a partial
profile
Both samples display heteroplasmy
at the same region
Consistent
One sample displays heteroplasmy;
one of these probe signals is also
present in the other sample
Consistent
One sample displays heteroplasmy;
the probe signals are not present in
the other sample
Difference
Sequencing: Reporting of Base Calls
1.
Sequence data should be determined from both complementary strands of DNA
for mtDNA regions HVI and HVII. Only under special circumstances (see 2.
below) can sequence be reported for confirmed data from a single-strand.
a.
All good quality data that shows concordance for both complementary
DNA strands or confirmed single-strand data can be reported. A list of
reported differences from the rCRS must be accompanied by the range of
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nucleotides of the region that was sequenced. All possible alternative
alignments are not reported.
2.
b.
For sequence where an ambiguous calling situation occurs for one strand,
it must be left unresolved and called an “N”. No more than 4 uneditable N calls are acceptable within any 10 base stretch of strand
sequence data.
c.
If an “N” base call is made on one of the DNA strands (eg., due to an
electrophoretic artifact), this base position can still be reported as a base in
the plurality consensus sequence as long as (i) the data on both strands are
not in conflict with each other, and (ii) the data generated from the
complementary or confirmatory DNA strand is clean and there is no
question regarding its base call.
d.
A minimum read length of 90 contiguous base pairs of double-stranded or
confirmed data that forms a contig will be valid for interpretation and for
generating weight assessment.
f.
A minimum read length of 90 consecutive bases of single-stranded data is
necessary for any strand to be used to build a contig. Only under special
circumstances (see 2. below) can data be reported for a read length of less
than 90 bases.
Special circumstances will arise (eg., length heteroplasmy) when data from only
one DNA strand can be obtained or read lengths of greater than 90 bases are not
possible.
a.
b.
For samples with HVI or HVII length heteroplasmy, additional primers
should be used in order to obtain as much complementary data as possible.
For sequence where no data is available for one of the complementary
strands, this can still be reported given that the sequencing reaction that
yielded the one strand of sequence data is repeated (confirmed) for this
sample with the same or different primer in the same direction. All of the
data from this region (eg., results from two cycle sequencing reactions)
must be concordant between the two sequencing runs. Note: This type of
rerun will satisfy conditions where a difference from rCRS or sequence
heteroplasmy is being reported.
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c.
3.
C.
D.
Situations will arise which result in severe length heteroplasmy (e.g. in
HVII, 310 C resulting in a homopolymeric stretch of 13 C residues).
Under these conditions, it will be not be possible to sequence through this
region in either the forward or reverse direction. This could result in the
trimming of a strand (e.g., C1) and/or will yield runs with sequences
generated from the complementary strand (e.g., D1) primer that are less
than 90 bases. In these cases, the data will be acceptable at less than 90
bases. The guidelines described in b. above for run confirmations will
also apply to the confirmation runs necessary in this scenario.
In situations when un-editable “N” base calls are made at a given sequence
position for both DNA strands, then this base will be reported as “N”. Samples
with 3 or more un-editable “N” calls within a 10 base pair region of the
consensus sequence in either HVI or HVII are inconclusive.
Criteria for Mixture Recognition
1.
More than two heteroplasmic positions in a sample are suggestive of a DNA
mixture. If possible, the sample should be re-extracted or other samples in the
same case should be tested.
2.
Samples that contain two heteroplasmic positions might warrant further testing of
additional samples depending on the circumstances of the case. This is to make
sure that the sample type in question is not due to a mixture.
Sequence Comparisons
1.
The positive control run with that sample must type correctly in order to report the
sequence for that sample.
2.
If either extraction or amplification negative controls contain readable sequences,
the associated case sample(s) must be compared to this data before any further
sequence comparisons are made. The readable extraction or amplification
negative controls must differ from all case samples by at least two bases for these
case samples to be interpreted and reported (see Control Tables, section A3).
3.
When comparing sequences obtained from samples, only the regions in common
will be considered.
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4.
A specimen that yields a mixture of DNA sequences is reported as inconclusive.
No comparisons and no statistical evaluation will be performed using this sample.
5.
The number of C nucleotides at the HVI polycytosine C-stretch will not be
considered for interpretation purposes if length heteroplasmy is present. Likewise,
the number of C residues exhibited in samples with HVII length heteroplasmy is
highly variable and care must be taken when making comparisons. In order for
sequence concordance to be declared, a common length variant must be observed
in both samples being compared.
Differences between samples due to the absence of an HVII common length
variant are treated as one difference.
6.
Match Criteria for Sequencing data
Concordance
Inconclusive
Exclusion
When two mtDNA sequences from separate samples (e.g. from
two pieces of evidence or from evidence and a maternal family
reference source) are consistent with each other in the
overlapping regions, the two samples cannot be excluded as
originating from the same person or from having a maternal
relationship, respectively.
The resulting comparison will be considered inconclusive
when two mtDNA sequences from separate samples differ by
one difference.
In these cases other reference sources and/or further testing in
order to obtain more sequence data may be helpful.
The resulting comparison will be considered an exclusion
when two mtDNA sequences from separate samples differ by
two or more differences.
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7.
Treatment of sequence heteroplasmy
Two identical heteroplasmic bases are present
at the same position in both samples.
This is not a difference (eg.,
C/T vs. C/T).
One heteroplasmic base is present in one
sample; a common base is present at the same
position in the other sample.
This is not a difference (eg.,
C/T vs. C; also C/T vs. T).
One heteroplasmic base is present in one
sample; a different base is present at the same
position in the other sample.
This is a difference (eg., C/T
vs. G).
Revision History:
July 24, 2010 – Initial version of procedure.
September 3, 2010 – Revised version of procedure: sentence removed from Paragraph Bb to reflect current procedures.
March 7, 2011 – General clarifications added; removed decision matrix for negative controls and added flow charts;
paragraph B.1 revised to reflect “a maximum number of 3 ‘N’ calls…”
March 17, 2012 – Changed current interpretation guideline regarding the number of “N” calls within 10 consecutive bases
from 3 to 4; Minor changes and typographical corrections.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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Statistical Analysis
The frequency of occurrence of evidence sample types will be reported based on the type of
analysis that was performed. When both Linear Array and DNA sequencing analysis are done
on a given comparison, only the DNA sequencing statistical analysis will be reported.
Nevertheless, the statistics for both analyses (when performed) will be included in the case file.
The extent of the sequence data that will be used for the database search and statistical analysis
will be limited to the shortest range and most conservative reporting of the sequence in common
between the evidence sample(s) and reference sample(s) used in the comparison (see previously
discussed sequence reporting criteria). Statistical analysis will not be performed on partial
Linear Array mitotypes.
Statistics may also be presented comparing evidentiary samples, in which case the statistical
analysis will be limited to the shortest range and most conservative reporting of the sequence in
common between the evidence samples.
A.
For Linear Array types (mitotypes), the laboratory will use a database containing
population sample mitotypes from Kline et al, 2005; complete database is found at:
http://www.cstl.nist.gov/biotech/strbase/NISTpopdata/NIST_mtDNA_LINEAR_ARRAY_data.xls
This population database contains mitotypes of 666 individuals and is comprised of the
following population groups: African-American (252), Caucasian (286), and Hispanic
(128).
1.
A search of this database can be done by using the LA Summary & Stats excel
spreadsheet located on the Forensic Biology network. After the spreadsheet is
opened, make sure the Sequence Output tab is selected. Then enter the Linear
Array mitotype at the top of the worksheet in this manner, with exceptions noted
on the stats spreadsheet:
a.
b.
c.
When no signal is seen in the Linear Array mitotype at a given
position, then a null type (“0”) is used for the database comparison at
that position.
When a weak signal is seen in the Linear Array mitotype, the
numerical type is used for the database comparison at that position.
This is a conservative search strategy.
When two signal types are seen in the Linear Array mitotype at a
given position, then each type is used separately for the database
search(es) and the resulting counts are combined. This is a
conservative search strategy.
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2.
B.
Click on the LA Stats tab to view the calculated population statistics for your
sample mitotype. The spreadsheet calculates frequency estimates for the mitotype
as described below (see B.3).
For sequencing data, use the database and the procedure suggested by the FBI.
1.
Database
The database used to obtain a frequency estimate is maintained by the FBI
(Budowle et al 1999, Monson et al 2002) and is available for download at the
following web address:
http://www.fbi.gov/hq/lab/fsc/backissu/april2002/miller1.htm.
A copy of the database including the search window is found on mtDNA analysts’
computers. The database contains HVI (16024-16365) and HVII (73-340)
sequences from a variety of unrelated individuals.
2.
Searching Profiles
The base pair range of the profile to be searched is limited to the shortest range of
reported sequence in common for both compared samples (see previously
discussed reporting criteria).
Click on the mtDNA icon on your screen. The search window will open. Several
options are pre-selected as indicated below.
Mode:
Database:
- search
- forensic
Under options (in edit menu):
Listing profiles:
- not checked
Length variants:
- consider multiple insertions as one difference
Partial profiles:
- not checked
Statistics:
- display up to 2 differences
Listing haplotypes:
- check to list haplotypes that appear multiple times
Date:
- check “all profiles”
Heteroplasmic scenarios:
- not checked
Helper Apps:
- not specified
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Enter your profile ID.
Enter the searchable basepair range for HVI and click Add. Repeat for HVII. If
your sample has the standard read length (see above) just double-click on the HVI
or HVII icons. Enter all differences from the rCRS.
Click search.
Select a temporary directory and name for the results file.
The search result consists of the number of samples with 0-2 mismatches to the
searched sample in the combined database and divided into different ethnic
groups.
ATTENTION:
When sequence heteroplasmy is present at a given position in the mtDNA
sequence, the mtDNA database will be searched with an “N” at that position.
Even though mtDNA sequence HVII polycytosine length variants are
entered, multiple C-stretch length variants at the same position are
considered as one difference during the database searches of concordant
sequences containing this region and will not add additional rarity. In
addition, the number of “C” residues in samples with HVI length
heteroplasmy is not considered for comparison purposes.
3.
Frequency estimate
a.
Frequency estimate when the Linear Array mitotype or mtDNA sequence
is observed at least once in database.
Raw frequency estimates for the occurrence of a given mtDNA profile
in the general population is based on the counting method as follows:
p = x/N (Eq. 1)
Where p is the frequency estimate; x is the number of times a profile has
been observed in the population database, and N is the number of profiles
in the population database.
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A confidence interval must be calculated from the results of the database
search in order to correct the counting results for sampling errors
according to the following equation
p ± 1.96 [(p) (1-p)/N]1/2 (Eq. 2)
The upper 95% confidence interval value (upper bound = p + 1.96 [(p) (1p)/N]1/2) is calculated as the maximum frequency of occurrence within
each population of the same mitotype or mtDNA sequence as the searched
profile.
The upper bound estimate can be calculated automatically using the
Popstats spreadsheet for sequencing statistics or the LA Summary and
Stats spreadsheet found on the Forensic Biology network drive.
Example #1: The Linear Array mitotype or mtDNA sequence is observed
3 times in a database containing 2000 sample profiles. The frequency
estimate is 3/2000 = 0.0015; the upper bound of the confidence interval is
equal to 0.0015 +1.96[(0.0015)(0.9985)/2000]1/2 = 0.0015 + 0.0017 =
0.0032.
Meaning of example #1: With 95% confidence, the maximum true
frequency of the mtDNA profile is 0.0032 or 0.32%, or 1 in 310. In other
words, at least 99.68% of the population can be excluded as the source of
the evidence.
b.
Frequency estimate when the Linear Array mitotype or mtDNA
sequence is not observed in the database.
The following equation is used:
1 - α 1/N (Eq. 3)
α is the confidence coefficient (use 0.05 for a 95% confidence interval),
and N is the number of individuals in the population.
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Example #2: The Linear Array mitotype or mtDNA sequence is observed
0 times in the database containing 2000 sample profiles. The frequency
estimate is 1-0.051/2000 = 1-0.999 = 0.001.
Meaning of example #2: For a database size of 2000 mitotypes or
sequence profiles, the frequency of a mtDNA profile not observed in the
database is 0.001 or 0.1%; or 1 in 1000, or, with 95% confidence, 99.9%
of the population can be excluded as being the source of the evidence.
c.
Based on the FBI database, the mtDNA population database search
software supplies separate results of the frequency estimates for four
major populations (African-American, Hispanic, Caucasian, and
Asian Origin). It is not the intent of the report to draw any inference
as to the population origin of the contributor(s) of the evidence.
d.
The Linear Array mitotype population database of Kline, et.al., 2005,
supplies separate results of the frequency for three major populations
(African-American, Caucasian, Hispanic). It is not the intent of the
report to draw any inference as to the population origin of the
contributor(s) of the evidence.
e.
Reports will present the upper bound 95% confidence interval
estimate for each population group, and express this as a percentage
and a frequency, e.g., an upper bound 95% confidence interval
estimate of 0.5% (1 in 200). Frequency estimate will be rounded
down to nearest 10 or single whole number. The intent of the report
is to present a conservative range of estimates of the strength of the
mitochondrial DNA comparison.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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CREATION OF A CASEFILE CD
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Creation of a Casefile CD
When all of the sequencing analysis is completed for a case, and upon request from members of
the court, a CD containing all of the data for that case may be created to fulfill the request.
1.
Insert a blank CD into the computer, and open the program “Roxio/Creator Home.”
Click on the “Data,” section and select “Data Disc.”
2.
Depending on the specific request, the levels of the disc will be created according to the
following:
CD Main Window
- “Evidence” File
- Analyzed 3130xl Run Files
- Analyzed 3130xl data files
- Sequence Analysis Report
- Sequencher Evidence Project File
- “Exemplars” File
- All analyzed 3130xl Run Files
- Analyzed 3130xl data files
- Sequence Analysis Report
- Sequencher Exemplar Project File
3.
Ensure that all 3130xl data is imported from the Analyzed Archive on the network, NOT
the superhero archive.
To ensure the quality of the disc, it is advisable to copy all of the necessary data to a
single location on the local computer that will create the CD. Once all of the
necessary files have been compiled in this file on the local hard drive, according to
the tree structure above, the entire contents of the local file can be added to the CD.
4.
For the “Volume Label” fill in the casefile number, (eg. FB05-0234).
5.
Label the new disk with the casefile number. The disk should be delivered to the Quality
Assurance Manager for transmittal.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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References
DNA Sequencing
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich
DP, Roe BA, Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization
of the human mitochondrial genome Nature 1981; 290: 457-465. MEDLINE- 81173052
PUBMED-7219534
Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis
and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet.
1999; 23 (2): 147. MEDLINE- 99438386 PUBMED- 10508508.
Applied Biosystems. 3130/3130xl Genetic Analyzers Getting Started Guide. 2004. Part
Number 4352715 Rev. B. Applied Biosystems, Foster City, CA.
Applied Biosystems. 3130/3130xl Genetic Analyzers Maintenance, Troubleshooting, and
Reference Guide. 2004. Part Number 4352716 Rev. B. Applied Biosystems, Foster City, CA.
Applied Biosystems. DNA Sequencing Analysis Software User Guide: Version 5.1 for
Windows® XP and 2000 Platforms. 2003. Part Number 4346366 Rev. B. Applied Biosystems,
Foster City, CA.
Budowle B, Fisher C, Polanskey D, Hartog BD, Kepler R, Elling J. Standardizing the
Nomenclature for mtDNA Haplotypes with an Intuitive Hierarchal Execution Software Program.
ISFG Conference. 2007.
Budowle B, Wilson MR, DiZinno JA, Stauffer C, Fasano MA, Holland MM, Monson KL.
Mitochondrial regions HVI and HVII population data. Forensic Sci Int 1999; 103: 23-35.
Carracedo A, Baer W, Lincoln P, Mayr W, Morling N, Olaisen B, Schneider P, Budowle B,
Brinkmann B, Gill P, Holland MM, Tully G, Wilson MR. DNA commission of the International
Society for Forensic Genetics: Guidelines for mitochondrial DNA typing. Forensic Sci Int 2000;
110: 79-85.
Gene Codes. Sequencher™ 4.0 User Manual. 1999. Gene Codes Corporation. Ann Arbor, MI.
Holland MM, Parsons TJ. Mitochondrial DNA sequence analysis - Validation and use for
forensic casework. Forensic Science Review 1999; 11: 21-50.
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Monson KL, Miller KWP, Wilson MR, DiZinno JA, Budowle B. The mtDNA population
database: An integrated software and database resource for forensic comparison, vol. 4, no. 2.
Forensic Science Communications 2002.
http://www.fbi.gov/hq/lab/fsc/backissu/april2002/miller1.htm
Sanger FS, Nilken S, Coulson AR. Sequencing with chain terminating inhibitors. Proc Nat Acad
Sci U.S.A. 1977; 74: 5463-5467.
Scientific Working Group on DNA Analysis Methods (SWGDAM).
Guidelines for
mitochondrial DNA (mtDNA) nucleotide sequence interpretation, vol. 5, no.2. Forensic Science
Communications 2003. http://www.fbi.gov/hq/lab/fsc/backissu/april2003/index.htm.
Stewart JEB, Aagaard PJ, Pokorak EG, Polanskey D, Budowle B. Evaluation of a multicapillary
electrophoresis instrument for mitochondrial DNA typing. J Forensic Sci 2003; 48: 571-580.
Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B. Validation of mitochondrial
DNA sequencing for forensic casework analysis. Int J Leg Med 1995; 108: 68-74.
Wilson MR, Allard MW, Monson KL, Miller KWP, Budowle B. Recommendations for
consistent treatment of length variants in the human mitochondrial DNA control region. Forensic
Sci Int 2002; 129: 35-42.
Wilson, MR., Allard, MW, Monson, KL, Miller KWP, Budowle B. Further discussion of the
consistent treatment of length variants in the human mitochondrial DNA control region, vol. 4,
no.4. Forensic Science Communications 2002.
http://www.fbi.gov/hq/lab/fsc/backissu/oct2002/index.htm.
ExoSAP-IT (PCR Product Clean-up)
Dugan, KA, Lawrence, HS, Hares, DR, Fisher, CL, Budowle, B. An improved method for postPCR purification for mtDNA sequence analysis. J Forensic Sci 2002; 47:811-18.
Product Gel
Roche Diagnostics Corp. DNA molecular weight marker XIV. Package Insert, Version 3.
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November 2002. Cat. No. 1 721 933. Roche Diagnostics Corporation, Indianapolis, IN.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 1989. Cold
Spring Harbor, NY.
Linear Array
Kline, M., Vallone, PM, Redman, JW, Duewer, DL, Calloway, CD, Butler, JM. Mitochondrial
DNA typing with control region and coding region SNPs. J. Forensic Sci. 2005; 50: 377- 385.
Roche Diagnostics Corp. Linear Array Mitochondrial DNA HVI/HVII Region-Sequence Typing
Kit. Package Insert, Version 3. September 2003. Cat. No. 03 527 867 001.
Agilent
Mueller, O, Hahnenberger K, Dittmann M, Yee H, Dubrow R, Nagle R, Ilsley D. A microfluidic
system for high-speed reproducible DNA sizing and quantitation. Electrophoresis 2000; 21:128134.
Mueller, O. High resolution DNA analysis with the DNA 500 and DNA 1000 LabChip® kits.
Agilent Technologies publication 5988-3041E 2001. www.agilent.com/chem
Jensen, M. Use of the Agilent 2100 Bioanalyzer and the DNA 500 LabChip in the Analysis of
PCR Amplified Mitochondrial DNA. Agilent Technologies publication 5989-0985EN 2004.
www.agilent.com/chem
Bjerketorp J, Chiang ANT, Hjort K, Rosenquist M, Liu W-T, Jansson JK. Rapid lab-on-a-chip
profiling of human gut bacteria. J. Microbiol. Methods 2008; 72: 82-90.
Agilent Bioanalyzer maintenance and troubleshooting guide, edition 11, 2003. Agilent
Technologies, Inc.
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APPENDIX A
OLIGONUCLEOTIDE PRIMER SEQUENCES
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Appendix A –Oglionucleotide Primer Sequences
Region
HVI
HVII
Primer
Nucleotide (base) Sequence
A11
5'- CAC CAT TAG CAC CCA AAG CT -3'
20
A4
5'- CCC CAT GCT TAC AAG CAA GT -3'
20
B1
5'- GAG GAT GGT GGT CAA GGG AC -3'
20
B4
5'- TTT GAT GTG GAT TGG GTT T -3'
19
HVIF2
5'- CTC CAC CAT TAG CAC CCA A -3'
19
HVIR
5'- ATT TCA CGG AGG ATG GTG -3'
18
C1
5'- CTC ACG GGA GCT CTC CAT GC -3'
20
C2
5'- TTA TTT ATC GCA CCT ACG TTC AAT -3'
24
D1
5'- CTG TTA AAA GTG CAT ACC GCC A -3'
22
D2
5'- GGG GTT TGG TGG AAA TTT TTT G -3'
22
HVIIF
5'- CAC CCT ATT AAC CAC TCA CG -3'
20
HVIIR
5'- CTG TTA AAA GTG CAT ACC GC -3'
20
Size (no. of bases)
1. Nucleotide sequences for primers A1, A4, B1, B4, C1, C2, D1, and D2 are from the FBI Laboratory
DNA Analysis Unit II Mitochondrial DNA Analysis Protocol (mtDNA Protocol Manual, DNA
Amplification - Rev. 8, Issue Date 02/01/05 for primers A1, B1, C1, C2, D1, and D2; mtDNA Protocol
Manual, Cycle Sequencing - Rev. 8, Issue Date 09/10/04 for primers A4 and B4). The primer sequences
in the FBI mtDNA Protocol Manual are based on those described in the following:
Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle, B. Validation of mitochondrial DNA
sequencing for forensic casework analysis. Int J of Leg Med 1995; 108:68-74.
Wilson MR, Polanskey D, Butler J, DiZinno JA, Replogle J, Budowle B. Extraction, PCR amplification,
and sequencing of mitochondrial DNA from human hair shafts, BioTechniques 1995; 18(4):662-669.
2. Nucleotide sequences for oligonucleotide primers HVIF, HVIR, HVIIF, HVIIR are from the product
insert for the LINEAR ARRAY Mitochondrial DNA HVI/HVII Region-Sequence Typing Kit that is
available from Roche Applied Sciences (Cat. No. 03-527-867-001; product information is available at
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OLIGONUCLEOTIDE PRIMER SEQUENCES
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www.roche-applied-science.com). The primer sequences in the typing kit are based on those described
in:
Gabriel MN, Calloway CD, Reynolds RL, Primorac D. Identification of human remains by immobilized
sequence-specific oligonucleotide probe analysis of mtDNA hepervariable regions I and II. Croat Med J
2003; 44:293-298.
Kline MC, Vallone PM, Redman JW, Duewer DL, Calloway CD, Butler JM. Mitochondrial DNA typing
screens with control region and coding region SNPs. J Forensic Sci 2005; 50:377-385.
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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APPENDIX B
MITOCHONDRIAL DNA PRIMER LOCATIONS
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340
73
16365
16024
Appendix B – Mitochondrial DNA Primer Locations
0
HVI – 342 bp
HVII – 268 bp
HVI Primers
HVII Primers
HVIF
A1
HVIIF
C1
A4
C2
B4
D2
D1
B1
HVIIR
HVIR
The above diagrams are not to scale. All primer positions are relative to the table below. All arrows
indicate the directions (forward or reverse) that the primer amplifies along the hypervariable region.
HVI (16024 - 16365) = 342 bp
HVII (73 - 340) = 268 bp
Position1
Primer
Position1
Primer
HVIF
15975
HVIIF
15
A1
15978
C1
29
A42
16190
C2
154
B42
16182
D23
306
B1
16410
D1
429
HVIR
16418
HVIIR
429
1
Nucleotide position is defined as the first base at the 5’ end of the primer.
2
Primers A4 and B4 are used to resolve C-stretch length polymorphisms in HVI.
3
Primer D2 is used when necessary to resolve the reverse strand sequence when C-stretch polymorphism is present in HVII.
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APPENDIX B
MITOCHONDRIAL DNA PRIMER LOCATIONS
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THIS PAGE INTENTIONALLY LEFT BLANK
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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APPENDIX C
REVISED CAMBRIDGE REFERENCE SEQUENCE
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Appendix C- Revised Cambridge Reference Sequence
Hypervariable Region I (HVI)
160241606416104161441618416224162641630416344-
TTCTTTCATG
TGACTCACCC
CTGCCAGCCA
TGACCACCTG
CCCCCTCCCC
TCAACTATCA
CACCCACTAG
TACATAGTAC
CAGTCAAATC
GGGAAGCAGA
ATCAACAACC
CCATGAATAT
TAGTACATAA
ATGCTTACAA
CACATCAACT
GATACCAACA
ATAAAGCCAT
CCTTCTCGTC
TTTGGGTACC ACCCAAGTAT
GCTATGTATT TCGTACATTA
TGTACGGTAC CATAAATACT
AAACCCAATC CACATCAAAA
GCAAGTACAG CAATCAACCC
GCAACTCCAA AGCCACCCCT
AACCTACCCA CCCTTAACAG
TTACCGTACA TAGCACATTA
CC -16365(end)
Hypervariable Region II (HVII)
73113153193233273313-
ATGCACGCGA
CCTATGTCGC
ATTATTTATC
ACTTACTAAA
TAATAATAAC
CAGACATCAT
CCCGCTTCTG
TAGCATTGCG
AGTATCTGTC
GCACCTACGT
GTGTGTTAAT
AATTGAATGT
AACAAAAAAT
GCCACAGCAC
AGACGCTGGA GCCGGAGCAC
TTTGATTCCT GCCTCATCCT
TCAATATTAC AGGCGAACAT
TAATTAATGC TTGTAGGACA
CTGCACAGCC ACTTTCCACA
TTCCACCAAA CCCCCCCTCC
TTAAACAC- 340(end)
Human Mitochondrial DNA Revised Cambridge Reference Sequence,
LOCUS NC_012920
16569 bp DNA
circular
PRI 30-APR-2010
DEFINITION
Homo sapiens mitochondrion, complete genome.
ACCESSION
NC_012920
AC_000021
VERSION
NC_012920.1
GI: 251831106
SOURCE
mitochondrion Homo sapiens (human)
ORGANISM
Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires;
Primates; Haplorrhini; Catarrhini; Hominidae; Homo.
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APPENDIX C
REVISED CAMBRIDGE REFERENCE SEQUENCE
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REFERENCES
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA,
Sanger F, Schreier PH, Smith AJ, Staden R, Young IG. Sequence and organization of the human mitochondrial
genome Nature 1981; 290: 457-465. MEDLINE- 81173052 PUBMED-7219534
Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the
Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 1999; 23 (2): 147. MEDLINE99438386 PUBMED- 10508508
Revision History:
July 24, 2010 – Initial version of procedure.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
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APPENDIX D
DETAILED CYCSEQ/3130XL CALCULATIONS
DATE EFFECTIVE
APPROVED BY
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07-16-2012
MITOCHONDRIAL DNA TECHNICAL LEADER
120 OF 121
Appendix D- Detailed CycSeq/3130xl Calculations
Example:
Sample
Vol,
Quant
(µL)
Amp. Neg
4
Sample
5
Num of
Vol, Linear
Vol,
Linear
Array (µl) Misc (µl)
Arrays
0
1
0
5.4
0
0
Cont,
Mean
HVIHVII
(ng/4 ul)
Vol, ExoSap (ul)
Vol, DNA
(µl)
Comment
Vol,
H2O
(µl)
0
9.2
3.0
Control
7.8
4.3
th
6.5
56
7.9
1/10 dil.
Calculations for Amplification Negative:
1.
A total of 4µL of the amplification negative was aliquotted for quantification. The quantification
value was below the limit of detection (nd) for that sample, and a linear array was not run.
Therefore, the analyst enters 4 into the “Vol, Quant” field and 0 each into the ”Num of Linear
Arrays”, “Col, Linear Array”, “Vol, Misc”, and “Conc, Mean HVI-HVII” fields.
2.
The total remaining reaction volume is calculated to be 46 µL (50 µL starting volume minus 4 µl
used for quantification).
3.
The amount of ExoSAP-IT required is calculated according to the following guideline: 1 µl of
ExoSAP-IT for every 5 µL of amplified product. Thus, the current reaction volume is divided by
5 (46/5) and 9.2 is automatically input into the “ExoSAP-IT” field.
4.
The amount of template to add to the cycle sequencing reaction is calculated. In this case, a quant
value of 0 instructs the program to enter the maximum volume amount of 3 µL into the template
field. The system automatically enters “Control” into the comments field based on the nd
quantification value.
5.
Finally, the amount of water is calculated based on the previously calculated sample volume to
make the total volume quantity sufficient at 10.8 µL. This DNA/water mixture is now ready to be
added to the cycle sequencing reaction.
Calculations for Sample:
1.
In this example, 5 µL of amplified sample was aliquotted for quantification. Also, 5.4 µL of the
sample was used for Linear Array analysis. The user also inputs 5 into the “Vol, Quant” field
along with the volume of 5.4 (µL) used for the Linear Array analysis into the “Vol, Linear Array”
field. The user inputs 0 into the “Vol, Misc” field. Finally, the user inputs the concentration
value into the “Conc, Mean HVI-HVII” field. This field is based on the amount of sample DNA
in a volume of 4 µL.
2.
The total reaction volume is calculated to be 39.6 µL, which is equal to the starting volume minus
5 µL for the quantification volume, 5.4 µL used for the Linear Array (50 - 5 - 5.4 = 39.6).
3.
The amount of ExoSAP-IT required is then determined as before. The calculation indicates that
the reaction requires 7.9 µL of ExoSAP-IT (39.6/5).
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Forensic Biology network. All printed versions are non-controlled copies.
© NYC OFFICE OF CHIEF MEDICAL EXAMINER
PROTOCOLS FOR FORENSIC MITOCHONDRIAL DNA ANALYSIS
APPENDIX D
DETAILED CYCSEQ/3130XL CALCULATIONS
4.
5.
DATE EFFECTIVE
APPROVED BY
PAGE
07-16-2012
MITOCHONDRIAL DNA TECHNICAL LEADER
121 OF 121
In calculating the amount of template required for the cycle sequencing reaction, the system first
determines the new concentration of the DNA sample after addition of ExoSAP-IT by the
dividing the original concentration [(56 ng/4 µL)(39.6 µL)] by the new volume (47.5 ul). The
new concentration (11.67 ng/ µL) is then used to calculate the volume of sample needed to equal
5 ng of sample DNA [(5 ng)/(11.67 ng/ µL) = 0.428 µL]. If the final volume is less than 1 µL,
the system will indicate that a dilution is necessary in the “Comment” filed. In this example, the
calculation indicates that 4.3 ul of a 1/10 sample dilution is required.
As before, the system indicates the amount of water necessary to yield a total of 10.8 µL of
sample volume for the next step of cycle sequencing.
Revision History:
July 24, 2010 – Initial version of procedure.
February 28, 2011 – Step 1 under “calculations for sample” modified.
July 16, 2012 – Minor revisions in content to generalize terminology for LIMS.
© NYC OFFICE OF CHIEF MEDICAL EXAMINER
Controlled versions of Department of Forensic Biology Documents only exist electronically on the
Forensic Biology network. All printed versions are non-controlled copies.